Compositions and methods for diagnosis and treating mood disorders

ABSTRACT

The present invention provides methods for diagnosing mental disorders such as mood disorders, including bipolar disorder I and II and major depression; The invention also provides methods of identifying modulators of such mental disorders as well as methods of using these modulators to treat patients suffering from such mental disorders.

CROSS-REFERENCES TO RELATED APPLICATIONS

The present application claims the benefit of U.S. Ser. No. 60/423,247,filed Nov. 1, 2002 and U.S. Ser. No. 60/431,454, filed Dec. 6, 2002, thedisclosures of which are hereby incorporated by reference in theirentirety for all purposes.

STATEMENT AS TO RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSOREDRESEARCH AND DEVELOPMENT

Not applicable.

BACKGROUND OF THE INVENTION

Clinical depression, including both bipolar disorders and majordepression disorders, is a major public health problem, affecting anestimated 9.5% of the adult population of the United States each year.While it has been hypothesized that mental disorders, including mooddisorders such as major depression and bipolar disorder as well aspsychotic disorders such as schizophrenia, have complex genetic roots,little progress has been made in identifying gene sequences and geneproducts that play a role in causing these disorders, as is true formany diseases with a complex genetic origin (see, e.g. Burmeister, Biol.Psychiatry 45:522-532 (1999)). Relying on the discovery that certaingenes expressed in particular brain pathways and regions are likelyinvolved in the development of mental disorders, the present inventionprovides methods for diagnosis and treatment of mental disorders, aswell as methods for identifying compounds effective in treating mentaldisorders.

BRIEF SUMMARY OF THE INVENTION

In order to further understand the neurobiology of mood disorders suchas bipolar disorders (BP) and major depression disorders (MDD), theinventors of the present application have used DNA microarrays to studyexpression profiles of human post-mortem brains from patients diagnosedwith BP or MDD. The work has focused on three brain regions: theanterior cingulated cortex (AnCg), the dorsolateral prefrontal cortex(DLPFC), and the cerebellum (CB).

The present invention demonstrates, for the first time, differentialexpression of the 72 nucleic acids listed in Table 2, the 16 nucleicacids listed in Table 3, or the 967 nucleic acids listed in Table 4, inthe brains of patients suffering from mood disorders, such as bipolardisorder and major depression disorder, in comparison with normalcontrol subjects. In addition, the present invention identifiesbiochemical pathways involved in mood disorders, where the proteinsencoded by the nucleic acids listed in Table 2, 3, or 4 are componentsof the biochemical pathways (e.g., the bFGF signal transduction pathway,the GPCR and cAMP/PI/Rho pathways, the proteasome pathway, the oxidativephosphorylation pathway, Myelination, Cytochrome P450, or the GABA andglutamate pathways; see also FIGS. 1-5, 10-13, and 15).

Finally, genes that are differentially expressed in MDD or BP and bygender are useful in diagnosing mood disorders, as the prevalence ofcertain mood disorders shows a gender bias. Differential expression bybrain region similarly is a useful diagnostic and therapeutic tool, ascertain mood disorders primarily affect certain brain regions.

This invention thus provides methods for determining whether a subjecthas or is predisposed for a mental disorder such as bipolar disorder ormajor depression disorder. The invention also provides methods ofproviding a prognosis and for monitoring disease progression andtreatment. Furthermore, the present invention provides nucleic acid andprotein targets for assays for drugs for the treatment of mentaldisorders such as bipolar disorder and major depression disorder.

In some embodiments, the methods comprise the steps of: (i) obtaining abiological sample from a subject; (ii) contacting the sample with areagent that selectively associates with a polynucleotide or polypeptideencoded by a nucleic acid that hybridizes under stringent conditions toa nucleotide sequence listed in Table 2, 3 or 4; and (iii) detecting thelevel of reagent that selectively associates with the sample, therebydetermining whether the subject has or is predisposed for a mentaldisorder.

In some embodiments, the reagent is an antibody. In some embodiments,the reagent is a nucleic acid. In some embodiments, the reagentassociates with a polynucleotide. In some embodiments, the reagentassociates with a polypeptide. In some embodiments, the polynucleotidecomprises a nucleotide sequence of a gene listed in Table 2, 3, or 4. Insome embodiment, the polypeptide comprises an amino acid sequence of agene listed in Table 2, 3, or 4. In some embodiments, the level ofreagent that associates with the sample is different (i.e., higher orlower) from a level associated with humans without a mental disorder. Insome embodiments, the biological sample is obtained from amniotic fluid.In some embodiments, the mental disorder is a mood disorder. In someembodiments, the mood disorder is selected from the group consisting ofbipolar disorder and major depression disorder.

The invention also provides methods of identifying a compound fortreatment of a mental disorder. In some embodiments, the methodscomprises the steps of: (i) contacting the compound with a polypeptide,which is encoded by a polynucleotide that hybridizes under stringentconditions to a nucleic acid comprising a nucleotide sequence of Table2, 3, or 4; and (ii) determining the functional effect of the compoundupon the polypeptide, thereby identifying a compound for treatment of amental disorder.

In some embodiments, the contacting step is performed in vitro. In someembodiment, the polypeptide comprises an amino acid sequence of a genelisted in Table 2, 3, or 4. In some embodiments, the polypeptide isexpressed in a cell or biological sample, and the cell or biologicalsample is contacted with the compound. In some embodiments, the mentaldisorder is a mood disorder or psychotic disorder. In some embodiments,the mood disorder is selected from the group consisting of bipolardisorder I and II and major depression. In some embodiments, thepsychotic disorder is schizophrenia. In some embodiments, the methodsfurther comprise administering the compound to an animal, e.g., ananimal subjected to stress as a model for depression and determining theeffect on the animal, e.g., an invertebrate, a vertebrate, or a mammal.In some embodiments, the determining step comprises testing the animal'smental function.

In some embodiments, the methods comprise the steps of (i) contactingthe compound to a cell, the cell comprising a polynucleotide thathybridizes under stringent conditions to a nucleotide sequence of Table2, 3, or 4; and (ii) selecting a compound that modulates expression ofthe polynucleotide, thereby identifying a compound for treatment of amental disorder. In some embodiments, the polynucleotide comprises anucleotide sequence listed in Table 2, 3, or 4. In some embodiment, theexpression of the polynucleotide is enhanced. In some embodiments, theexpression of the polynucleotide is decreased. In some embodiments, themethods further comprise administering the compound to an animal anddetermining the effect on the animal. In some embodiments, thedetermining step comprises testing the animal's mental function. In someembodiments, the mental disorder is a mood disorder or psychoticdisorder. In some embodiments, the mood disorder is selected from thegroup consisting of bipolar disorder I and II and major depression. Insome embodiments, the psychotic disorder is schizophrenia.

The invention also provides methods of treating a mental disorder in asubject. In some embodiments, the methods comprise the step ofadministering to the subject a therapeutically effective amount of acompound identified using the methods described above. In someembodiments, the mental disorder is a mood disorder or psychoticdisorder. In some embodiments, the mood disorder is selected from thegroup consisting of bipolar disorder I and II and major depression. Insome embodiments, the psychotic disorder is schizophrenia. In someembodiments, the compound is a small organic molecule, an antibody, anantisense molecule, aptamer, or a peptide.

The invention also provides methods of treating mental disorders in asubject, comprising the step of administering to the subject atherapeutically effective amount of a polypeptide, which is encoded by apolynucleotide that hybridizes under stringent conditions to a nucleicacid of Table 2, 3, or 4. In some embodiments, the polypeptide comprisesan amino acid sequence encoded by a gene listed in Table 2, 3, or 4. Insome embodiments, the mental disorder is a mood disorder or psychoticdisorder. In some embodiments, the psychotic disorder is schizophrenia.In some embodiments, the mood disorder is a bipolar disorder or majordepression.

The invention also provides methods of treating mental disorders in asubject, comprising the step of administering to the subject atherapeutically effective amount of a polynucleotide, which hybridizesunder stringent conditions to a nucleic acid of Table 2, 3, or 4. Insome embodiments, the mental disorder is a mood disorder or psychoticdisorder. In some embodiments, the psychotic disorder is schizophrenia.In some embodiments, the mood disorder is a bipolar disorder or majordepression.

BRIEF DESCRIPTION OF THE DRAWINGS

Table 1: Table 1 lists genes differentially expressed in mood disordersubjects.

Table 2: Table 2 lists 72 genes differentially expressed in mooddisorder subjects.

Table 3: Table 3 lists 16 genes differentially expressed in specificbrain regions and mood disorder.

Table 4: Table 4 lists 967 genes differentially expressed in mooddisorder subjects as determined by microarray analysis. Flag 1 indicatesthat the differential expression of the gene was confirmed by Real timePCR. Flag 2 indicates that differential expression of the gene wasconfirmed by anti-depressant studies. Flag 3 indicates that the genebelongs to an enriched gene ontology. Up and down indicates thedirection of the changes compared to controls.

Table 5: Table 5 lists Real time PCR results on sample genes that aredifferentially expressed in mood disorder subjects.

Table 6: Table 6 lists anti-depressant treatment results for genes thatare differentially expressed in mood disorder subjects.

Table 7: Tables 7A-D lists the gene ontology of selected genesdifferentially expressed in mood disorder subjects.

Table 8: Table 8 lists sample of genes that are differentially expressedin mood disorder subjects and are potential druggable targets.

FIG. 1 shows selected biochemical pathways for genes differentiallyexpressed in mood disorder subjects.

FIG. 2 summarizes functions for signal transduction transcriptsdifferentially expressed in MDD subjects.

FIG. 3 shows bFGF pathway transcripts differentially expressed in MDDsubjects.

FIG. 4 shows values for differential expression of bFGF transcripts inMDD subjects.

FIG. 5 shows selected biochemical pathways that are dysregulated in mooddisorders.

FIG. 6 shows selected biochemical pathways that are dysregulated in BPsubjects.

FIG. 7 shows three genes overexpressed in mood disorder subjects thatare located in the same chromosomal region.

FIG. 8 shows three genes overexpressed in mood disorder subjects thatare located on 15q11-13 in the Prader-Willi region.

FIG. 9 shows certain genes regulated in human postmortem tissue and byantidepressants in rats.

FIG. 10 shows selected biochemical pathways (i.e., the GPCR andcAMP/PI/Rho pathways) for genes differentially expressed in mooddisorder subjects. Two G protein coupled receptors, GPR37 and GPRC5B,are increased in both AnCg and DLPFC of BP patients, and decreased inMD. As downstream signaling pathways of GPCR, genes involved in cAMPpathway signaling are increased n BP patients, and decreased in MD.Genes involved in phosphatidylinositol pathways are deregulatedspecifically in MD.

FIG. 11 shows a selected biochemical pathway (i.e., the proteasomepathway) for genes differentially expressed in mood disorder subjects.The proteasome is an assembly of 28 alpha and beta subunits thatfunctions to degrade proteins. The proteasome is involved in regulationof protein turnover and in particular oxidized proteins. There is anover representation of proteasome genes found in cortical regions of BP,but not in the cerebellum, suggesting that some functional compensationin the proteasome is occurring in BP patients.

FIG. 12 shows a selected biochemical pathway (i.e., the oxidativephosphorylation pathway) for genes differentially expressed in mooddisorder subjects. The oxidative phosphorylation classification isinvolved in bioenergetics, metabolism, and as a byproduct can producereactive oxygen species. This pathway is overly expressed in bothbipolar and major depression, with differences between cortical regionsand cerebellum.

FIG. 13 shows an example of a growth factor system (e.g., FGF) that isaltered in mood disorders.

FIG. 14 shows RealTime PCR results which confirm that selectedFGF-related genes first identified using microarray analysis aredifferentially expressed in mood disorders.

FIG. 15 shows selected genes in biochemical pathways involving GABA andglutamate that are differentially expressed in mood disorder subjects.

DEFINITIONS

A “mental disorder” or “mental illness” or “mental disease” or“psychiatric or neuropsychiatric disease or illness or disorder” refersto mood disorders (e.g., major depression, mania, and bipolardisorders), psychotic disorders (e.g., schizophrenia, schizoaffectivedisorder, schizophreniform disorder, delusional disorder, briefpsychotic disorder, and shared psychotic disorder), personalitydisorders, anxiety disorders (e.g., obsessive-compulsive disorder) aswell as other mental disorders such as substance-related disorders,childhood disorders, dementia, autistic disorder, adjustment disorder,delirium, multi-infarct dementia, and Tourette's disorder as describedin Diagnostic and Statistical Manual of Mental Disorders, FourthEdition, (DSM IV). Typically, such disorders have a complex geneticand/or a biochemical component.

“A psychotic disorder” refers to a condition that affects the mind,resulting in at least some loss of contact with reality. Symptoms of apsychotic disorder include, e.g., hallucinations, changed behavior thatis not based on reality, delusions and the like. See, e.g., DSM IV.Schizophrenia, schizoaffective disorder, schizophreniform disorder,delusional disorder, brief psychotic disorder, substance-inducedpsychotic disorder, and shared psychotic disorder are examples ofpsychotic disorders.

“Schizophrenia” refers to a psychotic disorder involving a withdrawalfrom reality by an individual. Symptoms comprise for at least a part ofa month two or more of the following symptoms: delusions (only onesymptom is required if a delusion is bizarre, such as being abducted ina space ship from the sun); hallucinations (only one symptom is requiredif hallucinations are of at least two voices talking to one another orof a voice that keeps up a running commentary on the patient's thoughtsor actions); disorganized speech (e.g., frequent derailment orincoherence); grossly disorganized or catatonic behavior; or negativesymptoms, i.e., affective flattening, alogia, or avolition.Schizophrenia encompasses disorders such as, e.g., schizoaffectivedisorders. Diagnosis of schizophrenia is described in, e.g., DSM IV.Types of schizophrenia include, e.g., paranoid, disorganized, catatonic,undifferentiated, and residual.

A “mood disorder” refers to disruption of feeling tone or emotionalstate experienced by an individual for an extensive period of time. Mooddisorders include major depression disorder (i.e., unipolar disorder),mania, dysphoria, bipolar disorder, dysthymia, cyclothymia and manyothers. See, e.g., Diagnostic and Statistical Manual of MentalDisorders, Fourth Edition, (DSM IV).

“Major depression disorder,” “major depressive disorder,” or “unipolardisorder” refers to a mood disorder involving any of the followingsymptoms: persistent sad, anxious, or “empty” mood; feelings ofhopelessness or pessimism; feelings of guilt, worthlessness, orhelplessness; loss of interest or pleasure in hobbies and activitiesthat were once enjoyed, including sex; decreased energy, fatigue, being“slowed down”; difficulty concentrating, remembering, or makingdecisions; insomnia, early-morning awakening, or oversleeping; appetiteand/or weight loss or overeating and weight gain; thoughts of death orsuicide or suicide attempts; restlessness or irritability; or persistentphysical symptoms that do not respond to treatment, such as headaches,digestive disorders, and chronic pain. Various subtypes of depressionare described in, e.g., DSM IV.

“Bipolar disorder” is a mood disorder characterized by alternatingperiods of extreme moods. A person with bipolar disorder experiencescycling of moods that usually swing from being overly elated orirritable (mania) to sad and hopeless (depression) and then back again,with periods of normal mood in between. Diagnosis of bipolar disorder isdescribed in, e.g., DSM IV. Bipolar disorders include bipolar disorder I(mania with or without major depression) and bipolar disorder II(hypomania with major depression), see, e.g., DSM IV.

An “agonist” refers to an agent that binds to a polypeptide orpolynucleotide of the invention, stimulates, increases, activates,facilitates, enhances activation, sensitizes or up regulates theactivity or expression of a polypeptide or polynucleotide of theinvention.

An “antagonist” refers to an agent that inhibits expression of apolypeptide or polynucleotide of the invention or binds to, partially ortotally blocks stimulation, decreases, prevents, delays activation,inactivates, desensitizes, or down regulates the activity of apolypeptide or polynucleotide of the invention.

“Inhibitors,” “activators,” and “modulators” of expression or ofactivity are used to refer to inhibitory, activating, or modulatingmolecules, respectively, identified using in vitro and in vivo assaysfor expression or activity, e.g., ligands, agonists, antagonists, andtheir homologs and mimetics. The term “modulator” includes inhibitorsand activators. Inhibitors are agents that, e.g., inhibit expression ofa polypeptide or polynucleotide of the invention or bind to, partiallyor totally block stimulation or enzymatic activity, decrease, prevent,delay activation, inactivate, desensitize, or down regulate the activityof a polypeptide or polynucleotide of the invention, e.g., antagonists.Activators are agents that, e.g., induce or activate the expression of apolypeptide or polynucleotide of the invention or bind to, stimulate,increase, open, activate, facilitate, enhance activation or enzymaticactivity, sensitize or up regulate the activity of a polypeptide orpolynucleotide of the invention, e.g., agonists. Modulators includenaturally occurring and synthetic ligands, antagonists, agonists, smallchemical molecules and the like. Assays to identify inhibitors andactivators include, e.g., applying putative modulator compounds tocells, in the presence or absence of a polypeptide or polynucleotide ofthe invention and then determining the functional effects on apolypeptide or polynucleotide of the invention activity. Samples orassays comprising a polypeptide or polynucleotide of the invention thatare treated with a potential activator, inhibitor, or modulator arecompared to control samples without the inhibitor, activator, ormodulator to examine the extent of effect. Control samples (untreatedwith modulators) are assigned a relative activity value of 100%.Inhibition is achieved when the activity value of a polypeptide orpolynucleotide of the invention relative to the control is about 80%,optionally 50% or 25-1%. Activation is achieved when the activity valueof a polypeptide or polynucleotide of the invention relative to thecontrol is 110%, optionally 150%, optionally 200-500%, or 1000-3000%higher.

The term “test compound” or “drug candidate” or “modulator” orgrammatical equivalents as used herein describes any molecule, eithernaturally occurring or synthetic, e.g., protein, oligopeptide (e.g.,from about 5 to about 25 amino acids in length, preferably from about 10to 20 or 12 to 18 amino acids in length, preferably 12, 15, or 18 aminoacids in length), small organic molecule, polysaccharide, lipid, fattyacid, polynucleotide, oligonucleotide, etc. The test compound can be inthe form of a library of test compounds, such as a combinatorial orrandomized library that provides a sufficient range of diversity. Testcompounds are optionally linked to a fusion partner, e.g., targetingcompounds, rescue compounds, dimerization compounds, stabilizingcompounds, addressable compounds, and other functional moieties.Conventionally, new chemical entities with useful properties aregenerated by identifying a test compound (called a “lead compound”) withsome desirable property or activity, e.g., inhibiting activity, creatingvariants of the lead compound, and evaluating the property and activityof those variant compounds. Often, high throughput screening (HTS)methods are employed for such an analysis.

A “small organic molecule” refers to an organic molecule, eithernaturally occurring or synthetic, that has a molecular weight of morethan about 50 Daltons and less than about 2500 Daltons, preferably lessthan about 2000 Daltons, preferably between about 100 to about 1000Daltons, more preferably between about 200 to about 500 Daltons.

“Determining the functional effect” refers to assaying for a compoundthat increases or decreases a parameter that is indirectly or directlyunder the influence of a polynucleotide or polypeptide of the invention(such as a polynucleotide of Table 2, 3, or 4 or a polypeptide encodedby a gene of Table 2, 3, or 4), e.g., measuring physical and chemical orphenotypic effects. Such functional effects can be measured by any meansknown to those skilled in the art, e.g., changes in spectroscopic (e.g.,fluorescence, absorbance, refractive index), hydrodynamic (e.g., shape),chromatographic, or solubility properties for the protein; measuringinducible markers or transcriptional activation of the protein;measuring binding activity or binding assays, e.g. binding toantibodies; measuring changes in ligand binding affinity; measurement ofcalcium influx; measurement of the accumulation of an enzymatic productof a polypeptide of the invention or depletion of an substrate;measurement of changes in protein levels of a polypeptide of theinvention; measurement of RNA stability; G-protein binding; GPCRphosphorylation or dephosphorylation; signal transduction, e.g.,receptor-ligand interactions, second messenger concentrations (e.g.,cAMP, IP3, or intracellular Ca²⁺); identification of downstream orreporter gene expression (CAT, luciferase, β-gal, GFP and the like),e.g., via chemiluminescence, fluorescence, calorimetric reactions,antibody binding, inducible markers, and ligand binding assays.

Samples or assays comprising a nucleic acid or protein disclosed hereinthat are treated with a potential activator, inhibitor, or modulator arecompared to control samples without the inhibitor, activator, ormodulator to examine the extent of inhibition. Control samples(untreated with inhibitors) are assigned a relative protein activityvalue of 100%. Inhibition is achieved when the activity value relativeto the control is about 80%, preferably 50%, more preferably 25-0%.Activation is achieved when the activity value relative to the control(untreated with activators) is 110%, more preferably 150%, morepreferably 200-500% (i.e., two to five fold higher relative to thecontrol), more preferably 1000-3000% higher.

“Biological sample” includes sections of tissues such as biopsy andautopsy samples, and frozen sections taken for histologic purposes. Suchsamples include blood, spinal fluid, sputum, tissue, lysed cells, brainbiopsy, cultured cells, e.g., primary cultures, explants, andtransformed cells, stool, urine, etc. A biological sample is typicallyobtained from a eukaryotic organism, most preferably a mammal such as aprimate, e.g., chimpanzee or human; cow; dog; cat; a rodent, e.g.,guinea pig, rat, mouse; rabbit; or a bird; reptile; or fish.

“Antibody” refers to a polypeptide substantially encoded by animmunoglobulin gene or immunoglobulin genes, or fragments thereof whichspecifically bind and recognize an analyte (antigen). The recognizedimmunoglobulin genes include the kappa, lambda, alpha, gamma, delta,epsilon and mu constant region genes, as well as the myriadimmunoglobulin variable region genes. Light chains are classified aseither kappa or lambda. Heavy chains are classified as gamma, mu, alpha,delta, or epsilon, which in turn define the immunoglobulin classes, IgG,IgM, IgA, IgD and IgE, respectively.

An exemplary immunoglobulin (antibody) structural unit comprises atetramer. Each tetramer is composed of two identical pairs ofpolypeptide chains, each pair having one “light” (about 25 kD) and one“heavy” chain (about 50-70 kD). The N-terminus of each chain defines avariable region of about 100 to 110 or more amino acids primarilyresponsible for antigen recognition. The terms variable light chain(V_(L)) and variable heavy chain (V_(H)) refer to these light and heavychains respectively.

Antibodies exist, e.g., as intact immunoglobulins or as a number ofwell-characterized fragments produced by digestion with variouspeptidases. Thus, for example, pepsin digests an antibody below thedisulfide linkages in the hinge region to produce F(ab)′₂, a dimer ofFab which itself is a light chain joined to V_(H)—C_(H)1 by a disulfidebond. The F(ab)′₂ may be reduced under mild conditions to break thedisulfide linkage in the hinge region, thereby converting the F(ab)′₂dimer into an Fab′ monomer. The Fab′ monomer is essentially an Fab withpart of the hinge region (see, Paul (Ed.) Fundamental Immunology, ThirdEdition, Raven Press, NY (1993)). While various antibody fragments aredefined in terms of the digestion of an intact antibody, one of skillwill appreciate that such fragments may be synthesized de novo eitherchemically or by utilizing recombinant DNA methodology. Thus, the termantibody, as used herein, also includes antibody fragments eitherproduced by the modification of whole antibodies or those synthesized denovo using recombinant DNA methodologies (e.g., single chain Fv).

The terms “peptidomimetic” and “mimetic” refer to a synthetic chemicalcompound that has substantially the same structural and functionalcharacteristics of the polynucleotides, polypeptides, antagonists oragonists of the invention. Peptide analogs are commonly used in thepharmaceutical industry as non-peptide drugs with properties analogousto those of the template peptide. These types of non-peptide compoundare termed “peptide mimetics” or “peptidomimetics” (Fauchere, Adv. DrugRes. 15:29 (1986); Veber and Freidinger TINS p. 392 (1985); and Evans etal., J. Med. Chem. 30:1229 (1987), which are incorporated herein byreference). Peptide mimetics that are structurally similar totherapeutically useful peptides may be used to produce an equivalent orenhanced therapeutic or prophylactic effect. Generally, peptidomimeticsare structurally similar to a paradigm polypeptide (i.e., a polypeptidethat has a biological or pharmacological activity), such as a CCX CKR,but have one or more peptide linkages optionally replaced by a linkageselected from the group consisting of, e.g., —CH₂NH—, —CH₂S—, —CH₂—CH₂—,—CH═CH— (cis and trans), —COCH₂—, —CH(OH)CH₂—, and —CH₂SO—. The mimeticcan be either entirely composed of synthetic, non-natural analogues ofamino acids, or, is a chimeric molecule of partly natural peptide aminoacids and partly non-natural analogs of amino acids. The mimetic canalso incorporate any amount of natural amino acid conservativesubstitutions as long as such substitutions also do not substantiallyalter the mimetic's structure and/or activity. For example, a mimeticcomposition is within the scope of the invention if it is capable ofcarrying out the binding or enzymatic activities of a polypeptide orpolynucleotide of the invention or inhibiting or increasing theenzymatic activity or expression of a polypeptide or polynucleotide ofthe invention.

The term “gene” means the segment of DNA involved in producing apolypeptide chain; it includes regions preceding and following thecoding region (leader and trailer) as well as intervening sequences(introns) between individual coding segments (exons).

The term “isolated,” when applied to a nucleic acid or protein, denotesthat the nucleic acid or protein is essentially free of other cellularcomponents with which it is associated in the natural state. It ispreferably in a homogeneous state although it can be in either a dry oraqueous solution. Purity and homogeneity are typically determined usinganalytical chemistry techniques such as polyacrylamide gelelectrophoresis or high performance liquid chromatography. A proteinthat is the predominant species present in a preparation issubstantially purified. In particular, an isolated gene is separatedfrom open reading frames that flank the gene and encode a protein otherthan the gene of interest. The term “purified” denotes that a nucleicacid or protein gives rise to essentially one band in an electrophoreticgel. Particularly, it means that the nucleic acid or protein is at least85% pure, more preferably at least 95% pure, and most preferably atleast 99% pure.

The term “nucleic acid” or “polynucleotide” refers todeoxyribonucleotides or ribonucleotides and polymers thereof in eithersingle- or double-stranded form. Unless specifically limited, the termencompasses nucleic acids containing known analogues of naturalnucleotides that have similar binding properties as the referencenucleic acid and are metabolized in a manner similar to naturallyoccurring nucleotides. Unless otherwise indicated, a particular nucleicacid sequence also implicitly encompasses conservatively modifiedvariants thereof (e.g., degenerate codon substitutions), alleles,orthologs, SNPs (haplotypes), and complementary sequences as well as thesequence explicitly indicated. Specifically, degenerate codonsubstitutions may be achieved by generating sequences in which the thirdposition of one or more selected (or all) codons is substituted withmixed-base and/or deoxyinosine residues (Batzer et al., Nucleic AcidRes. 19:5081 (1991); Ohtsuka et al., J. Biol. Chem. 260:2605-2608(1985); and Cassol et al. (1992); Rossolini et al., Mol. Cell. Probes8:91-98 (1994)). The term nucleic acid is used interchangeably withgene, cDNA, and mRNA encoded by a gene.

The terms “polypeptide,” “peptide,” and “protein” are usedinterchangeably herein to refer to a polymer of amino acid residues. Theterms apply to amino acid polymers in which one or more amino acidresidue is an artificial chemical mimetic of a corresponding naturallyoccurring amino acid, as well as to naturally occurring amino acidpolymers and non-naturally occurring amino acid polymers. As usedherein, the terms encompass amino acid chains of any length, includingfull-length proteins (i.e., antigens), wherein the amino acid residuesare linked by covalent peptide bonds.

The term “amino acid” refers to naturally occurring and synthetic aminoacids, as well as amino acid analogs and amino acid mimetics thatfunction in a manner similar to the naturally occurring amino acids.Naturally occurring amino acids are those encoded by the genetic code,as well as those amino acids that are later modified, e.g.,hydroxyproline, γ-carboxyglutamate, and O-phosphoserine. Amino acidanalogs refers to compounds that have the same basic chemical structureas a naturally occurring amino acid, i.e., an cl carbon that is bound toa hydrogen, a carboxyl group, an amino group, and an R group, e.g.,homoserine, norleucine, methionine sulfoxide, methionine methylsulfonium. Such analogs have modified R groups (e.g., norleucine) ormodified peptide backbones, but retain the same basic chemical structureas a naturally occurring amino acid. “Amino acid mimetics” refers tochemical compounds that have a structure that is different from thegeneral chemical structure of an amino acid, but that functions in amanner similar to a naturally occurring amino acid.

Amino acids may be referred to herein by either the commonly known threeletter symbols or by the one-letter symbols recommended by the IUPAC-IUBBiochemical Nomenclature Commission. Nucleotides, likewise, may bereferred to by their commonly accepted single-letter codes.

“Conservatively modified variants” applies to both amino acid andnucleic acid sequences. With respect to particular nucleic acidsequences, “conservatively modified variants” refers to those nucleicacids that encode identical or essentially identical amino acidsequences, or where the nucleic acid does not encode an amino acidsequence, to essentially identical sequences. Because of the degeneracyof the genetic code, a large number of functionally identical nucleicacids encode any given protein. For instance, the codons GCA, GCC, GCGand GCU all encode the amino acid alanine. Thus, at every position wherean alanine is specified by a codon, the codon can be altered to any ofthe corresponding codons described without altering the encodedpolypeptide. Such nucleic acid variations are “silent variations,” whichare one species of conservatively modified variations. Every nucleicacid sequence herein that encodes a polypeptide also describes everypossible silent variation of the nucleic acid. One of skill willrecognize that each codon in a nucleic acid (except AUG, which isordinarily the only codon for methionine, and TGG, which is ordinarilythe only codon for tryptophan) can be modified to yield a functionallyidentical molecule. Accordingly, each silent variation of a nucleic acidthat encodes a polypeptide is implicit in each described sequence.

As to amino acid sequences, one of skill will recognize that individualsubstitutions, deletions or additions to a nucleic acid, peptide,polypeptide, or protein sequence which alters, adds or deletes a singleamino acid or a small percentage of amino acids in the encoded sequenceis a “conservatively modified variant” where the alteration results inthe substitution of an amino acid with a chemically similar amino acid.Conservative substitution tables providing functionally similar aminoacids are well known in the art. Such conservatively modified variantsare in addition to and do not exclude polymorphic variants, interspecieshomologs, and alleles of the invention.

The following eight groups each contain amino acids that areconservative substitutions for one another:

1) Alanine (A), Glycine (G);

2) Aspartic acid (D), Glutamic acid (E);

3) Asparagine (N), Glutamine (Q); 4) Arginine (R), Lysine (K); 5)Isoleucine (I), Leucine (L), Methionine (M), Valine (V); 6)Phenylalanine (F), Tyrosine (Y), Tryptophan (W); 7) Serine (S),Threonine (T); and 8) Cysteine (C), Methionine (M)

(see, e.g., Creighton, Proteins (1984)).

“Percentage of sequence identity” is determined by comparing twooptimally aligned sequences over a comparison window, wherein theportion of the polynucleotide sequence in the comparison window maycomprise additions or deletions (i.e., gaps) as compared to thereference sequence (which does not comprise additions or deletions) foroptimal alignment of the two sequences. The percentage is calculated bydetermining the number of positions at which the identical nucleic acidbase or amino acid residue occurs in both sequences to yield the numberof matched positions, dividing the number of matched positions by thetotal number of positions in the window of comparison and multiplyingthe result by 100 to yield the percentage of sequence identity.

The terms “identical” or percent “identity,” in the context of two ormore nucleic acids or polypeptide sequences, refer to two or moresequences or subsequences that are the same or have a specifiedpercentage of amino acid residues or nucleotides that are the same(i.e., 60% identity, optionally 65%, 70%, 75%, 80%, 85%, 90%, or 95%identity over a specified region), when compared and aligned for maximumcorrespondence over a comparison window, or designated region asmeasured using one of the following sequence comparison algorithms or bymanual alignment and visual inspection. Such sequences are then said tobe “substantially identical.” This definition also refers to thecomplement of a test sequence. Optionally, the identity exists over aregion that is at least about 50 nucleotides in length, or morepreferably over a region that is 100 to 500 or 1000 or more nucleotidesin length.

For sequence comparison, typically one sequence acts as a referencesequence, to which test sequences are compared. When using a sequencecomparison algorithm, test and reference sequences are entered into acomputer, subsequence coordinates are designated, if necessary, andsequence algorithm program parameters are designated. Default programparameters can be used, or alternative parameters can be designated. Thesequence comparison algorithm then calculates the percent sequenceidentities for the test sequences relative to the reference sequence,based on the program parameters.

A “comparison window”, as used herein, includes reference to a segmentof any one of the number of contiguous positions selected from the groupconsisting of from 20 to 600, usually about 50 to about 200, moreusually about 100 to about 150 in which a sequence may be compared to areference sequence of the same number of contiguous positions after thetwo sequences are optimally aligned. Methods of alignment of sequencesfor comparison are well known in the art. Optimal alignment of sequencesfor comparison can be conducted, e.g., by the local homology algorithmof Smith and Waterman (1970) Adv. Appl. Math. 2:482c, by the homologyalignment algorithm of Needleman and Wunsch (1970) J. Mol. Biol. 48:443,by the search for similarity method of Pearson and Lipman (1988) Proc.Nat.'l. Acad. Sci. USA 85:2444, by computerized implementations of thesealgorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin GeneticsSoftware Package, Genetics Computer Group, 575 Science Dr., Madison,Wis.), or by manual alignment and visual inspection (see, e.g., Ausubelet al., Current Protocols in Molecular Biology (1995 supplement)).

An example of an algorithm that is suitable for determining percentsequence identity and sequence similarity are the BLAST and BLAST 2.0algorithms, which are described in Altschul et al. (1977) Nuc. AcidsRes. 25:3389-3402, and Altschul et al. (1990) J. Mol. Biol. 215:403-410,respectively. Software for performing BLAST analyses is publiclyavailable through the National Center for Biotechnology Information.This algorithm involves first identifying high scoring sequence pairs(HSPs) by identifying short words of length W in the query sequence,which either match or satisfy some positive-valued threshold score Twhen aligned with a word of the same length in a database sequence. T isreferred to as the neighborhood word score threshold (Altschul et al.,supra). These initial neighborhood word hits act as seeds for initiatingsearches to find longer HSPs containing them. The word hits are extendedin both directions along each sequence for as far as the cumulativealignment score can be increased. Cumulative scores are calculatedusing, for nucleotide sequences, the parameters M (reward score for apair of matching residues; always >0) and N (penalty score formismatching residues; always <0). For amino acid sequences, a scoringmatrix is used to calculate the cumulative score. Extension of the wordhits in each direction are halted when: the cumulative alignment scorefalls off by the quantity X from its maximum achieved value; thecumulative score goes to zero or below, due to the accumulation of oneor more negative-scoring residue alignments; or the end of eithersequence is reached. The BLAST algorithm parameters W, T, and Xdetermine the sensitivity and speed of the alignment. The BLASTN program(for nucleotide sequences) uses as defaults a wordlength (W) of 11, anexpectation (E) or 10, M=5, N=−4 and a comparison of both strands. Foramino acid sequences, the BLASTP program uses as defaults a wordlengthof 3, and expectation (E) of 10, and the BLOSUM62 scoring matrix (seeHenikoff and Henikoff (1989) Proc. Natl. Acad. Sci. USA 89:10915)alignments (B) of 50, expectation (E) of 10, M=5, N=−4, and a comparisonof both strands.

The BLAST algorithm also performs a statistical analysis of thesimilarity between two sequences (see, e.g., Karlin and Altschul (1993)Proc. Natl. Acad. Sci. USA 90:5873-5787). One measure of similarityprovided by the BLAST algorithm is the smallest sum probability (P(N)),which provides an indication of the probability by which a match betweentwo nucleotide or amino acid sequences would occur by chance. Forexample, a nucleic acid is considered similar to a reference sequence ifthe smallest sum probability in a comparison of the test nucleic acid tothe reference nucleic acid is less than about 0.2, more preferably lessthan about 0.01, and most preferably less than about 0.001.

An indication that two nucleic acid sequences or polypeptides aresubstantially identical is that the polypeptide encoded by the firstnucleic acid is immunologically cross reactive with the antibodiesraised against the polypeptide encoded by the second nucleic acid, asdescribed below. Thus, a polypeptide is typically substantiallyidentical to a second polypeptide, for example, where the two peptidesdiffer only by conservative substitutions. Another indication that twonucleic acid sequences are substantially identical is that the twomolecules or their complements hybridize to each other under stringentconditions, as described below. Yet another indication that two nucleicacid sequences are substantially identical is that the same primers canbe used to amplify the sequence.

The phrase “selectively (or specifically) hybridizes to” refers to thebinding, duplexing, or hybridizing of a molecule only to a particularnucleotide sequence under stringent hybridization conditions when thatsequence is present in a complex mixture (e.g., total cellular orlibrary DNA or RNA).

The phrase “stringent hybridization conditions” refers to conditionsunder which a probe will hybridize to its target subsequence, typicallyin a complex mixture of nucleic acid, but to no other sequences.Stringent conditions are sequence-dependent and will be different indifferent circumstances. Longer sequences hybridize specifically athigher temperatures. An extensive guide to the hybridization of nucleicacids is found in Tijssen, Techniques in Biochemistry and MolecularBiology—Hybridization with Nucleic Probes, “Overview of principles ofhybridization and the strategy of nucleic acid assays” (1993).Generally, stringent conditions are selected to be about 5-10° C. lowerthan the thermal melting point (T_(m)) for the specific sequence at adefined ionic strength pH. The T_(m) is the temperature (under definedionic strength, pH, and nucleic concentration) at which 50% of theprobes complementary to the target hybridize to the target sequence atequilibrium (as the target sequences are present in excess, at T_(m),50% of the probes are occupied at equilibrium). Stringent conditionswill be those in which the salt concentration is less than about 1.0 Msodium ion, typically about 0.01 to 1.0 M sodium ion concentration (orother salts) at pH 7.0 to 8.3 and the temperature is at least about 30°C. for short probes (e.g., 10 to 50 nucleotides) and at least about 60°C. for long probes (e.g., greater than 50 nucleotides). Stringentconditions may also be achieved with the addition of destabilizingagents such as formamide. For selective or specific hybridization, apositive signal is at least two times background, optionally 10 timesbackground hybridization. Exemplary stringent hybridization conditionscan be as following: 50% formamide, 5×SSC, and 1% SDS, incubating at 42°C., or 5×SSC, 1% SDS, incubating at 65° C., with wash in 0.2×SSC, and0.1% SDS at 65° C. Such washes can be performed for 5, 15, 30, 60, 120,or more minutes. Nucleic acids that hybridize to the genes listed inTables 1-8 are encompassed by the invention.

Nucleic acids that do not hybridize to each other under stringentconditions are still substantially identical if the polypeptides thatthey encode are substantially identical. This occurs, for example, whena copy of a nucleic acid is created using the maximum codon degeneracypermitted by the genetic code. In such cases, the nucleic acidstypically hybridize under moderately stringent hybridization conditions.Exemplary “moderately stringent hybridization conditions” include ahybridization in a buffer of 40% formamide, 1 M NaCl, 1% SDS at 37° C.,and a wash in 1×SSC at 45° C. Such washes can be performed for 5, 15,30, 60, 120, or more minutes. A positive hybridization is at least twicebackground. Those of ordinary skill will readily recognize thatalternative hybridization and wash conditions can be utilized to provideconditions of similar stringency.

For PCR, a temperature of about 36° C. is typical for low stringencyamplification, although annealing temperatures may vary between about32° C. and 48° C. depending on primer length. For high stringency PCRamplification, a temperature of about 62° C. is typical, although highstringency annealing temperatures can range from about 50° C. to about65° C., depending on the primer length and specificity. Typical cycleconditions for both high and low stringency amplifications include adenaturation phase of 90° C.-95° C. for 30 sec-2 min., an annealingphase lasting 30 sec.-2 min., and an extension phase of about 72° C. for1-2 min. Protocols and guidelines for low and high stringencyamplification reactions are provided, e.g., in Innis et al., PCRProtocols, A Guide to Methods and Applications (1990).

The phrase “a nucleic acid sequence encoding” refers to a nucleic acidthat contains sequence information for a structural RNA such as rRNA, atRNA, or the primary amino acid sequence of a specific protein orpeptide, or a binding site for a trans-acting regulatory agent. Thisphrase specifically encompasses degenerate codons (i.e., differentcodons which encode a single amino acid) of the native sequence orsequences which may be introduced to conform with codon preference in aspecific host cell.

The term “recombinant” when used with reference, e.g., to a cell, ornucleic acid, protein, or vector, indicates that the cell, nucleic acid,protein or vector, has been modified by the introduction of aheterologous nucleic acid or protein or the alteration of a nativenucleic acid or protein, or that the cell is derived from a cell somodified. Thus, for example, recombinant cells express genes that arenot found within the native (nonrecombinant) form of the cell or expressnative genes that are otherwise abnormally expressed, under-expressed ornot expressed at all.

The term “heterologous” when used with reference to portions of anucleic acid indicates that the nucleic acid comprises two or moresubsequences that are not found in the same relationship to each otherin nature. For instance, the nucleic acid is typically recombinantlyproduced, having two or more sequences from unrelated genes arranged tomake a new functional nucleic acid, e.g., a promoter from one source anda coding region from another source. Similarly, a heterologous proteinindicates that the protein comprises two or more subsequences that arenot found in the same relationship to each other in nature (e.g., afusion protein).

An “expression vector” is a nucleic acid construct, generatedrecombinantly or synthetically, with a series of specified nucleic acidelements that permit transcription of a particular nucleic acid in ahost cell. The expression vector can be part of a plasmid, virus, ornucleic acid fragment. Typically, the expression vector includes anucleic acid to be transcribed operably linked to a promoter.

The phrase “specifically (or selectively) binds to an antibody” or“specifically (or selectively) immunoreactive with”, when referring to aprotein or peptide, refers to a binding reaction which is determinativeof the presence of the protein in the presence of a heterogeneouspopulation of proteins and other biologics. Thus, under designatedimmunoassay conditions, the specified antibodies bind to a particularprotein and do not bind in a significant amount to other proteinspresent in the sample. Specific binding to an antibody under suchconditions may require an antibody that is selected for its specificityfor a particular protein. For example, antibodies raised against aprotein having an amino acid sequence encoded by any of thepolynucleotides of the invention can be selected to obtain antibodiesspecifically immunoreactive with that protein and not with otherproteins, except for polymorphic variants. A variety of immunoassayformats may be used to select antibodies specifically immunoreactivewith a particular protein. For example, solid-phase ELISA immunoassays,Western blots, or immunohistochemistry are routinely used to selectmonoclonal antibodies specifically immunoreactive with a protein. See,Harlow and Lane Antibodies, A Laboratory Manual, Cold Spring HarborPublications, NY (1988) for a description of immunoassay formats andconditions that can be used to determine specific immunoreactivity.Typically, a specific or selective reaction will be at least twice thebackground signal or noise and more typically more than 10 to 100 timesbackground.

One who is “predisposed for a mental disorder” as used herein means aperson who has an inclination or a higher likelihood of developing amental disorder when compared to an average person in the generalpopulation.

DETAILED DESCRIPTION OF THE INVENTION I. Introduction

To understand the complex genetic basis of mental disorders, the presentinvention provides studies that have been conducted to investigate theexpression patterns of genes that are differentially expressedspecifically in central nervous system of subjects with mood disorders.The large spectrum of symptoms associated with mental disorders islikely a reflection of the complex genetic basis and complex geneexpression patterns in patients with mental disorders. Differentcombinations of the genes disclosed herein can be responsible for one ormore mental disorders. Furthermore, brain pathways or circuits as wellas subcellular pathways are important for understanding the developmentand diagnosis of mental disorders. The selected brain regions describedherein (AnCng, DLPFC, and CB) are implicated in the clinical symptoms ofmental disorders such as mood disorders. Brain imaging studies focusingon particular brain regions, cytoarchitectural changes in brain regions,expression of key neurotransmitters or related molecules in brainregions, and subcellular pathways in brain regions all contribute to thedevelopment of mental disorders, and thus are an important considerationin the diagnosis and therapeutic uses described herein.

The present invention demonstrates the altered expression (either higheror lower expression) of the genes of Tables 1-8 at the mRNA level inselected brain regions of patients diagnosed with mood disorders (e.g.,bipolar disorder and major depression disorder) in comparison withnormal individuals. This invention thus provides methods for diagnosisof mental disorders such as mood disorders (e.g., bipolar disorder,major depression, and the like), psychotic disorders (e.g.,schizophrenia, and the like), and other mental disorders by detectingthe level of a transcript or translation product of the genes listed inTables 1-8 as well as their corresponding biochemical pathways. Thechromosomal location of such genes can be used to discover other genesin the region that are linked to development of a particular disorder.

The invention further provides methods of identifying a compound usefulfor the treatment of such disorders by selecting compounds thatmodulates the functional effect of the translation products or theexpression of the transcripts described herein. The invention alsoprovides for methods of treating patients with such mental disorders,e.g., by administering the compounds of the invention or by genetherapy.

The genes and the polypeptides that they encode, which are associatedwith mood disorders such as bipolar disease and major depression, areuseful for facilitating the design and development of various moleculardiagnostic tools such as GeneChip™ containing probe sets specific forall or selected mental disorders, including but not limited to mooddisorders, and as an ante- and/or post-natal diagnostic tool forscreening newborns in concert with genetic counseling. Other diagnosticapplications include evaluation of disease susceptibility, prognosis,and monitoring of disease or treatment process, as well as providingindividualized medicine via predictive drug profiling systems, e.g., bycorrelating specific genomic motifs with the clinical response of apatient to individual drugs. In addition, the present invention isuseful for multiplex SNP or haplotype profiling, including but notlimited to the identification of pharmacogenetic targets at the gene,mRNA, protein, and pathway level.

The genes and the polypeptides that they encode, described herein, asalso useful as drug targets for the development of therapeutic drugs forthe treatment or prevention of mental disorders, including but notlimited to mood disorders. Mental disorders have a high co-morbiditywith other neurological disorders, such as Parkinson's disease orAlzheimer's. Therefore, the present invention can be used for diagnosisand treatment of patients with multiple disease states that include amental disorder such as a mood disorder.

For example, antidepressants belong to different classes, e.g.,desipramine, bupropion, and fluoxetine are in general equally effect forthe treatment of clinical depression, but act by different mechanisms.The similar effectiveness of the drugs for treatment of mood disorderssuggests that they act through a yet as unidentified common pathway. Wedisclose herein that different classes of antidepressants (specificserotonin reuptake inhibitors, like fluoxetine and tricyclicantidepressants, like desipramine) regulate a common gene, and/or acommon group of genes as well as a unique set of genes when the humanand animal results herein are compared.

II. General Recombinant Nucleic Acid Methods for Use with the Invention

In numerous embodiments of the present invention, polynucleotides of theinvention will be isolated and cloned using recombinant methods. Suchpolynucleotides include, e.g., those listed in Tables 1-8, which can beused for, e.g., protein expression or during the generation of variants,derivatives, expression cassettes, to monitor gene expression, for theisolation or detection of sequences of the invention in differentspecies, for diagnostic purposes in a patient, e.g., to detect mutationsor to detect expression levels of nucleic acids or polypeptides of theinvention. In some embodiments, the sequences of the invention areoperably linked to a heterologous promoter. In one embodiment, thenucleic acids of the invention are from any mammal, including, inparticular, e.g., a human, a mouse, a rat, a primate, etc.

A. General Recombinant Nucleic Acids Methods

This invention relies on routine techniques in the field of recombinantgenetics. Basic texts disclosing the general methods of use in thisinvention include Sambrook et al., Molecular Cloning, A LaboratoryManual (3rd ed. 2001); Kriegler, Gene Transfer and Expression. ALaboratory Manual (1990); and Current Protocols in Molecular Biology(Ausubel et al., eds., 1994)).

For nucleic acids, sizes are given in either kilobases (kb) or basepairs (bp). These are estimates derived from agarose or acrylamide gelelectrophoresis, from sequenced nucleic acids, or from published DNAsequences. For proteins, sizes are given in kilodaltons (kDa) or aminoacid residue numbers. Proteins sizes are estimated from gelelectrophoresis, from sequenced proteins, from derived amino acidsequences, or from published protein sequences.

Oligonucleotides that are not commercially available can be chemicallysynthesized according to the solid phase phosphoramidite triester methodfirst described by Beaucage & Caruthers, Tetrahedron Letts. 22:1859-1862(1981), using an automated synthesizer, as described in Van Devanter et.al., Nucleic Acids Res. 12:6159-6168 (1984). Purification ofoligonucleotides is by either native acrylamide gel electrophoresis orby anion-exchange HPLC as described in Pearson & Reanier, J. Chrom.255:137-149 (1983).

The sequence of the cloned genes and synthetic oligonucleotides can beverified after cloning using, e.g., the chain termination method forsequencing double-stranded templates of Wallace et al., Gene 16:21-26(1981).

B. Cloning Methods for the Isolation of Nucleotide Sequences EncodingDesired Proteins

In general, the nucleic acids encoding the subject proteins are clonedfrom DNA sequence libraries that are made to encode cDNA or genomic DNA.The particular sequences can be located by hybridizing with anoligonucleotide probe, the sequence of which can be derived from thesequences of the genes listed in Tables 1-8, which provide a referencefor PCR primers and defines suitable regions for isolating specificprobes. Alternatively, where the sequence is cloned into an expressionlibrary, the expressed recombinant protein can be detectedimmunologically with antisera or purified antibodies made against apolypeptide comprising an amino acid sequence encoded by a gene listedin Table 1-8.

Methods for making and screening genomic and cDNA libraries are wellknown to those of skill in the art (see, e.g., Gubler and Hoffman Gene25:263-269 (1983); Benton and Davis Science, 196:180-182 (1977); andSambrook, supra). Brain cells are an example of suitable cells toisolate RNA and cDNA sequences of the invention.

Briefly, to make the cDNA library, one should choose a source that isrich in mRNA. The mRNA can then be made into cDNA, ligated into arecombinant vector, and transfected into a recombinant host forpropagation, screening and cloning. For a genomic library, the DNA isextracted from a suitable tissue and either mechanically sheared orenzymatically digested to yield fragments of preferably about 5-100 kb.The fragments are then separated by gradient centrifugation fromundesired sizes and are constructed in bacteriophage lambda vectors.These vectors and phage are packaged in vitro, and the recombinantphages are analyzed by plaque hybridization. Colony hybridization iscarried out as generally described in Grunstein et al., Proc. Natl.Acad. Sci. USA., 72:3961-3965 (1975).

An alternative method combines the use of synthetic oligonucleotideprimers with polymerase extension on an mRNA or DNA template. Suitableprimers can be designed from specific sequences of the invention. Thispolymerase chain reaction (PCR) method amplifies the nucleic acidsencoding the protein of interest directly from mRNA, cDNA, genomiclibraries or cDNA libraries. Restriction endonuclease sites can beincorporated into the primers. Polymerase chain reaction or other invitro amplification methods may also be useful, for example, to clonenucleic acids encoding specific proteins and express said proteins, tosynthesize nucleic acids that will be used as probes for detecting thepresence of mRNA encoding a polypeptide of the invention inphysiological samples, for nucleic acid sequencing, or for otherpurposes (see, U.S. Pat. Nos. 4,683,195 and 4,683,202). Genes amplifiedby a PCR reaction can be purified from agarose gels and cloned into anappropriate vector.

Appropriate primers and probes for identifying polynucleotides of theinvention from mammalian tissues can be derived from the sequencesprovided herein. For a general overview of PCR, see, Innis et al. PCRProtocols. A Guide to Methods and Applications, Academic Press, SanDiego (1990).

Synthetic oligonucleotides can be used to construct genes. This is doneusing a series of overlapping oligonucleotides, usually 40-120 bp inlength, representing both the sense and anti-sense strands of the gene.These DNA fragments are then annealed, ligated and cloned.

A gene encoding a polypeptide of the invention can be cloned usingintermediate vectors before transformation into mammalian cells forexpression. These intermediate vectors are typically prokaryote vectorsor shuttle vectors. The proteins can be expressed in either prokaryotes,using standard methods well known to those of skill in the art, oreukaryotes as described infra.

III. Purification of Proteins of the Invention

Either naturally occurring or recombinant polypeptides of the inventioncan be purified for use in functional assays. Naturally occurringpolypeptides, e.g., polypeptides encoded by genes listed in Tables 1-8,can be purified, for example, from mouse or human tissue such as brainor any other source of an ortholog. Recombinant polypeptides can bepurified from any suitable expression system.

The polypeptides of the invention may be purified to substantial purityby standard techniques, including selective precipitation with suchsubstances as ammonium sulfate; column chromatography,immunopurification methods, and others (see, e.g., Scopes, ProteinPurification Principles and Practice (1982); U.S. Pat. No. 4,673,641;Ausubel et al., supra; and Sambrook et al., supra).

A number of procedures can be employed when recombinant polypeptides arepurified. For example, proteins having established molecular adhesionproperties can be reversible fused to polypeptides of the invention.With the appropriate ligand, the polypeptides can be selectivelyadsorbed to a purification column and then freed from the column in arelatively pure form. The fused protein is then removed by enzymaticactivity. Finally the polypeptide can be purified using immunoaffinitycolumns.

A. Purification of Proteins from Recombinant Bacteria

When recombinant proteins are expressed by the transformed bacteria inlarge amounts, typically after promoter induction, although expressioncan be constitutive, the proteins may form insoluble aggregates. Thereare several protocols that are suitable for purification of proteininclusion bodies. For example, purification of aggregate proteins(hereinafter referred to as inclusion bodies) typically involves theextraction, separation and/or purification of inclusion bodies bydisruption of bacterial cells typically, but not limited to, byincubation in a buffer of about 100-150 μg/ml lysozyme and 0.1% NonidetP40, a non-ionic detergent. The cell suspension can be ground using aPolytron grinder (Brinkman Instruments, Westbury, N.Y.). Alternatively,the cells can be sonicated on ice. Alternate methods of lysing bacteriaare described in Ausubel et al. and Sambrook et al., both supra, andwill be apparent to those of skill in the art.

The cell suspension is generally centrifuged and the pellet containingthe inclusion bodies resuspended in buffer which does not dissolve butwashes the inclusion bodies, e.g., 20 mM Tris-HCl (pH 7.2), 1 mM EDTA,150 mM NaCl and 2% Triton-X 100, a non-ionic detergent. It may benecessary to repeat the wash step to remove as much cellular debris aspossible. The remaining pellet of inclusion bodies may be resuspended inan appropriate buffer (e.g., 20 mM sodium phosphate, pH 6.8, 150 mMNaCl). Other appropriate buffers will be apparent to those of skill inthe art.

Following the washing step, the inclusion bodies are solubilized by theaddition of a solvent that is both a strong hydrogen acceptor and astrong hydrogen donor (or a combination of solvents each having one ofthese properties). The proteins that formed the inclusion bodies maythen be renatured by dilution or dialysis with a compatible buffer.Suitable solvents include, but are not limited to, urea (from about 4 Mto about 8 M), formamide (at least about 80%, volume/volume basis), andguanidine hydrochloride (from about 4 M to about 8 M). Some solventsthat are capable of solubilizing aggregate-forming proteins, such as SDS(sodium dodecyl sulfate) and 70% formic acid, are inappropriate for usein this procedure due to the possibility of irreversible denaturation ofthe proteins, accompanied by a lack of immunogenicity and/or activity.Although guanidine hydrochloride and similar agents are denaturants,this denaturation is not irreversible and renaturation may occur uponremoval (by dialysis, for example) or dilution of the denaturant,allowing re-formation of the immunologically and/or biologically activeprotein of interest. After solubilization, the protein can be separatedfrom other bacterial proteins by standard separation techniques.

Alternatively, it is possible to purify proteins from bacteriaperiplasm. Where the protein is exported into the periplasm of thebacteria, the periplasmic fraction of the bacteria can be isolated bycold osmotic shock in addition to other methods known to those of skillin the art (see, Ausubel et al., supra). To isolate recombinant proteinsfrom the periplasm, the bacterial cells are centrifuged to form apellet. The pellet is resuspended in a buffer containing 20% sucrose. Tolyse the cells, the bacteria are centrifuged and the pellet isresuspended in ice-cold 5 mM MgSO₄ and kept in an ice bath forapproximately 10 minutes. The cell suspension is centrifuged and thesupernatant decanted and saved. The recombinant proteins present in thesupernatant can be separated from the host proteins by standardseparation techniques well known to those of skill in the art.

B. Standard Protein Separation Techniques For Purifying Proteins 1.Solubility Fractionation

Often as an initial step, and if the protein mixture is complex, aninitial salt fractionation can separate many of the unwanted host cellproteins (or proteins derived from the cell culture media) from therecombinant protein of interest. The preferred salt is ammonium sulfate.Ammonium sulfate precipitates proteins by effectively reducing theamount of water in the protein mixture. Proteins then precipitate on thebasis of their solubility. The more hydrophobic a protein is, the morelikely it is to precipitate at lower ammonium sulfate concentrations. Atypical protocol is to add saturated ammonium sulfate to a proteinsolution so that the resultant ammonium sulfate concentration is between20-30%. This will precipitate the most hydrophobic proteins. Theprecipitate is discarded (unless the protein of interest is hydrophobic)and ammonium sulfate is added to the supernatant to a concentrationknown to precipitate the protein of interest. The precipitate is thensolubilized in buffer and the excess salt removed if necessary, througheither dialysis or diafiltration. Other methods that rely on solubilityof proteins, such as cold ethanol precipitation, are well known to thoseof skill in the art and can be used to fractionate complex proteinmixtures.

2. Size Differential Filtration

Based on a calculated molecular weight, a protein of greater and lessersize can be isolated using ultrafiltration through membranes ofdifferent pore sizes (for example, Amicon or Millipore membranes). As afirst step, the protein mixture is ultrafiltered through a membrane witha pore size that has a lower molecular weight cut-off than the molecularweight of the protein of interest. The retentate of the ultrafiltrationis then ultrafiltered against a membrane with a molecular cut offgreater than the molecular weight of the protein of interest. Therecombinant protein will pass through the membrane into the filtrate.The filtrate can then be chromatographed as described below.

3. Column Chromatography

The proteins of interest can also be separated from other proteins onthe basis of their size, net surface charge, hydrophobicity and affinityfor ligands. In addition, antibodies raised against proteins can beconjugated to column matrices and the proteins immunopurified. All ofthese methods are well known in the art.

It will be apparent to one of skill that chromatographic techniques canbe performed at any scale and using equipment from many differentmanufacturers (e.g., Pharmacia Biotech).

IV. Detection of Gene Expression

Those of skill in the art will recognize that detection of expression ofpolynucleotides of the invention has many uses. For example, asdiscussed herein, detection of the level of polypeptides orpolynucleotides of the invention in a patient is useful for diagnosingmental disorders including mood disorders or psychotic disorders or apredisposition for a mood disorder or psychotic disorder. Moreover,detection of gene expression is useful to identify modulators ofexpression of the polypeptides or polynucleotides of the invention.

A variety of methods of specific DNA and RNA measurement using nucleicacid hybridization techniques are known to those of skill in the art(see, Sambrook, supra). Some methods involve an electrophoreticseparation (e.g., Southern blot for detecting DNA, and Northern blot fordetecting RNA), but measurement of DNA and RNA can also be carried outin the absence of electrophoretic separation (e.g., by dot blot).Southern blot of genomic DNA (e.g., from a human) can be used forscreening for restriction fragment length polymorphism (RFLP) to detectthe presence of a genetic disorder affecting a polypeptide of theinvention.

The selection of a nucleic acid hybridization format is not critical. Avariety of nucleic acid hybridization formats are known to those skilledin the art. For example, common formats include sandwich assays andcompetition or displacement assays. Hybridization techniques aregenerally described in Hames and Higgins Nucleic Acid Hybridization, APractical Approach, IRL Press (1985); Gall and Pardue, Proc. Natl. Acad.Sci. U.S.A., 63:378-383 (1969); and John et al. Nature, 223:582-587(1969).

Detection of a hybridization complex may require the binding of asignal-generating complex to a duplex of target and probepolynucleotides or nucleic acids. Typically, such binding occurs throughligand and anti-ligand interactions as between a ligand-conjugated probeand an anti-ligand conjugated with a signal. The binding of the signalgeneration complex is also readily amenable to accelerations by exposureto ultrasonic energy.

The label may also allow indirect detection of the hybridizationcomplex. For example, where the label is a hapten or antigen, the samplecan be detected by using antibodies. In these systems, a signal isgenerated by attaching fluorescent or enzyme molecules to the antibodiesor in some cases, by attachment to a radioactive label (see, e.g.,Tijssen, “Practice and Theory of Enzyme Immunoassays,” LaboratoryTechniques in Biochemistry and Molecular Biology, Burdon and vanKnippenberg Eds., Elsevier (1985), pp. 9-20).

The probes are typically labeled either directly, as with isotopes,chromophores, lumiphores, chromogens, or indirectly, such as withbiotin, to which a streptavidin complex may later bind. Thus, thedetectable labels used in the assays of the present invention can beprimary labels (where the label comprises an element that is detecteddirectly or that produces a directly detectable element) or secondarylabels (where the detected label binds to a primary label, e.g., as iscommon in immunological labeling). Typically, labeled signal nucleicacids are used to detect hybridization. Complementary nucleic acids orsignal nucleic acids may be labeled by any one of several methodstypically used to detect the presence of hybridized polynucleotides. Themost common method of detection is the use of autoradiography with ³H,¹²⁵I, ³⁵S, ¹⁴C, or ³²P-labeled probes or the like.

Other labels include, e.g., ligands that bind to labeled antibodies,fluorophores, chemiluminescent agents, enzymes, and antibodies which canserve as specific binding pair members for a labeled ligand. Anintroduction to labels, labeling procedures and detection of labels isfound in Polak and Van Noorden Introduction to Immunocytochemistry, 2nded., Springer Verlag, NY (1997); and in Haugland Handbook of FluorescentProbes and Research Chemicals, a combined handbook and cataloguePublished by Molecular Probes, Inc. (1996).

In general, a detector which monitors a particular probe or probecombination is used to detect the detection reagent label. Typicaldetectors include spectrophotometers, phototubes and photodiodes,microscopes, scintillation counters, cameras, film and the like, as wellas combinations thereof. Examples of suitable detectors are widelyavailable from a variety of commercial sources known to persons of skillin the art. Commonly, an optical image of a substrate comprising boundlabeling moieties is digitized for subsequent computer analysis.

Most typically, the amount of RNA is measured by quantifying the amountof label fixed to the solid support by binding of the detection reagent.Typically, the presence of a modulator during incubation will increaseor decrease the amount of label fixed to the solid support relative to acontrol incubation which does not comprise the modulator, or as comparedto a baseline established for a particular reaction type. Means ofdetecting and quantifying labels are well known to those of skill in theart.

In preferred embodiments, the target nucleic acid or the probe isimmobilized on a solid support. Solid supports suitable for use in theassays of the invention are known to those of skill in the art. As usedherein, a solid support is a matrix of material in a substantially fixedarrangement.

A variety of automated solid-phase assay techniques are alsoappropriate. For instance, very large scale immobilized polymer arrays(VLSIPS™), available from Affymetrix, Inc. (Santa Clara, Calif.) can beused to detect changes in expression levels of a plurality of genesinvolved in the same regulatory pathways simultaneously. See, Tijssen,supra., Fodor et al. (1991) Science, 251: 767-777; Sheldon et al. (1993)Clinical Chemistry 39(4): 718-719, and Kozal et al. (1996) NatureMedicine 2(7): 753-759.

Detection can be accomplished, for example, by using a labeled detectionmoiety that binds specifically to duplex nucleic acids (e.g., anantibody that is specific for RNA-DNA duplexes). One preferred exampleuses an antibody that recognizes DNA-RNA heteroduplexes in which theantibody is linked to an enzyme (typically by recombinant or covalentchemical bonding). The antibody is detected when the enzyme reacts withits substrate, producing a detectable product. Coutlee et al. (1989)Analytical Biochemistry 181:153-162; Bogulavski (1986) et al. J.Immunol. Methods 89:123-130; Prooijen-Knegt (1982) Exp. Cell Res.141:397-407; Rudkin (1976) Nature 265:472-473, Stollar (1970) Proc.Nat'l Acad. Sci. USA 65:993-1000; Ballard (1982) Mol. Immunol.19:793-799; Pisetsky and Caster (1982) Mol. Immunol. 19:645-650; Viscidiet al. (1988) J. Clin. Microbial. 41:199-209; and Kiney et al. (1989) J.Clin. Microbiol. 27:6-12 describe antibodies to RNA duplexes, includinghomo and heteroduplexes. Kits comprising antibodies specific for DNA:RNAhybrids are available, e.g., from Digene Diagnostics, Inc. (Beltsville,Md.).

In addition to available antibodies, one of skill in the art can easilymake antibodies specific for nucleic acid duplexes using existingtechniques, or modify those antibodies that are commercially or publiclyavailable. In addition to the art referenced above, general methods forproducing polyclonal and monoclonal antibodies are known to those ofskill in the art (see, e.g., Paul (3rd ed.) Fundamental Immunology RavenPress, Ltd., NY (1993); Coligan Current Protocols in ImmunologyWiley/Greene, NY (1991); Harlow and Lane Antibodies: A Laboratory ManualCold Spring Harbor Press, NY (1988); Stites et al. (eds.) Basic andClinical Immunology (4th ed.) Lange Medical Publications, Los Altos,Calif., and references cited therein; Goding Monoclonal Antibodies:Principles and Practice (2d ed.) Academic Press, New York, N.Y., (1986);and Kohler and Milstein Nature 256: 495-497 (1975)). Other suitabletechniques for antibody preparation include selection of libraries ofrecombinant antibodies in phage or similar vectors (see, Huse et al.Science 246:1275-1281 (1989); and Ward et al. Nature 341:544-546(1989)). Specific monoclonal and polyclonal antibodies and antisera willusually bind with a K_(D) of at least about 0.1 μM, preferably at leastabout 0.01 μM or better, and most typically and preferably, 0.001 μM orbetter.

The nucleic acids used in this invention can be either positive ornegative probes. Positive probes bind to their targets and the presenceof duplex formation is evidence of the presence of the target. Negativeprobes fail to bind to the suspect target and the absence of duplexformation is evidence of the presence of the target. For example, theuse of a wild type specific nucleic acid probe or PCR primers may serveas a negative probe in an assay sample where only the nucleotidesequence of interest is present.

The sensitivity of the hybridization assays may be enhanced through useof a nucleic acid amplification system that multiplies the targetnucleic acid being detected. Examples of such systems include thepolymerase chain reaction (PCR) system, in particular RT-PCR or realtime PCR, and the ligase chain reaction (LCR) system. Other methodsrecently described in the art are the nucleic acid sequence basedamplification (NASBA, Cangene, Mississauga, Ontario) and Q BetaReplicase systems. These systems can be used to directly identifymutants where the PCR or LCR primers are designed to be extended orligated only when a selected sequence is present. Alternatively, theselected sequences can be generally amplified using, for example,nonspecific PCR primers and the amplified target region later probed fora specific sequence indicative of a mutation.

An alternative means for determining the level of expression of thenucleic acids of the present invention is in situ hybridization. In situhybridization assays are well known and are generally described inAngerer et al., Methods Enzymol. 152:649-660 (1987). In an in situhybridization assay, cells or tissue, preferentially human cells ortissue from the cerebellum or the hippocampus, are fixed to a solidsupport, typically a glass slide. If DNA is to be probed, the cells aredenatured with heat or alkali. The cells are then contacted with ahybridization solution at a moderate temperature to permit annealing ofspecific probes that are labeled. The probes are preferably labeled withradioisotopes or fluorescent reporters.

V. Immunological Detection of the Polypeptides of the Invention

In addition to the detection of polynucleotide expression using nucleicacid hybridization technology, one can also use immunoassays to detectpolypeptides of the invention. Immunoassays can be used to qualitativelyor quantitatively analyze polypeptides. A general overview of theapplicable technology can be found in Harlow & Lane, Antibodies. ALaboratory Manual (1988).

A. Antibodies to Target Polypeptides or Other Immunogens

Methods for producing polyclonal and monoclonal antibodies that reactspecifically with a protein of interest or other immunogen are known tothose of skill in the art (see, e.g., Coligan, supra; and Harlow andLane, supra; Stites et al., supra and references cited therein; Goding,supra; and Kohler and Milstein Nature, 256:495-497 (1975)). Suchtechniques include antibody preparation by selection of antibodies fromlibraries of recombinant antibodies in phage or similar vectors (see,Huse et al., supra; and Ward et al., supra). For example, in order toproduce antisera for use in an immunoassay, the protein of interest oran antigenic fragment thereof, is isolated as described herein. Forexample, a recombinant protein is produced in a transformed cell line.An inbred strain of mice or rabbits is immunized with the protein usinga standard adjuvant, such as Freund's adjuvant, and a standardimmunization protocol. Alternatively, a synthetic peptide derived fromthe sequences disclosed herein and conjugated to a carrier protein canbe used as an immunogen.

Polyclonal sera are collected and titered against the immunogen in animmunoassay, for example, a solid phase immunoassay with the immunogenimmobilized on a solid support. Polyclonal antisera with a titer of 10⁴or greater are selected and tested for their cross-reactivity againstunrelated proteins or even other homologous proteins from otherorganisms, using a competitive binding immunoassay. Specific monoclonaland polyclonal antibodies and antisera will usually bind with a K_(D) ofat least about 0.1 mM, more usually at least about 1 μM, preferably atleast about 0.1 μM or better, and most preferably, 0.01 μM or better.

A number of proteins of the invention comprising immunogens may be usedto produce antibodies specifically or selectively reactive with theproteins of interest. Recombinant protein is the preferred immunogen forthe production of monoclonal or polyclonal antibodies. Naturallyoccurring protein, such as one comprising an amino acid sequence encodedby a gene listed in Table 1-8 may also be used either in pure or impureform. Synthetic peptides made using the protein sequences describedherein may also be used as an immunogen for the production of antibodiesto the protein. Recombinant protein can be expressed in eukaryotic orprokaryotic cells and purified as generally described supra. The productis then injected into an animal capable of producing antibodies. Eithermonoclonal or polyclonal antibodies may be generated for subsequent usein immunoassays to measure the protein.

Methods of production of polyclonal antibodies are known to those ofskill in the art. In brief, an immunogen, preferably a purified protein,is mixed with an adjuvant and animals are immunized. The animal's immuneresponse to the immunogen preparation is monitored by taking test bleedsand determining the titer of reactivity to the polypeptide of interest.When appropriately high titers of antibody to the immunogen areobtained, blood is collected from the animal and antisera are prepared.Further fractionation of the antisera to enrich for antibodies reactiveto the protein can be done if desired (see, Harlow and Lane, supra).

Monoclonal antibodies may be obtained using various techniques familiarto those of skill in the art. Typically, spleen cells from an animalimmunized with a desired antigen are immortalized, commonly by fusionwith a myeloma cell (see, Kohler and Milstein, Eur. J. Immunol.6:511-519 (1976)). Alternative methods of immortalization include, e.g.,transformation with Epstein Barr Virus, oncogenes, or retroviruses, orother methods well known in the art. Colonies arising from singleimmortalized cells are screened for production of antibodies of thedesired specificity and affinity for the antigen, and yield of themonoclonal antibodies produced by such cells may be enhanced by varioustechniques, including injection into the peritoneal cavity of avertebrate host. Alternatively, one may isolate DNA sequences whichencode a monoclonal antibody or a binding fragment thereof by screeninga DNA library from human B cells according to the general protocoloutlined by Huse et al., supra.

Once target protein specific antibodies are available, the protein canbe measured by a variety of immunoassay methods with qualitative andquantitative results available to the clinician. For a review ofimmunological and immunoassay procedures in general see, Stites, supra.Moreover, the immunoassays of the present invention can be performed inany of several configurations, which are reviewed extensively in MaggioEnzyme Immunoassay, CRC Press, Boca Raton, Fla. (1980); Tijssen, supra;and Harlow and Lane, supra.

Immunoassays to measure target proteins in a human sample may use apolyclonal antiserum that was raised to the protein (e.g., one has anamino acid sequence encoded by a gene listed in Table 1-8) or a fragmentthereof. This antiserum is selected to have low cross-reactivity againstdifferent proteins and any such cross-reactivity is removed byimmunoabsorption prior to use in the immunoassay.

B. Immunological Binding Assays

In a preferred embodiment, a protein of interest is detected and/orquantified using any of a number of well-known immunological bindingassays (see, e.g., U.S. Pat. Nos. 4,366,241; 4,376,110; 4,517,288; and4,837,168). For a review of the general immunoassays, see also AsaiMethods in Cell Biology Volume 37: Antibodies in Cell Biology, AcademicPress, Inc. NY (1993); Stites, supra. Immunological binding assays (orimmunoassays) typically utilize a “capture agent” to specifically bindto and often immobilize the analyte (in this case a polypeptide of thepresent invention or antigenic subsequences thereof). The capture agentis a moiety that specifically binds to the analyte. In a preferredembodiment, the capture agent is an antibody that specifically binds,for example, a polypeptide of the invention. The antibody may beproduced by any of a number of means well known to those of skill in theart and as described above.

Immunoassays also often utilize a labeling agent to specifically bind toand label the binding complex formed by the capture agent and theanalyte. The labeling agent may itself be one of the moieties comprisingthe antibody/analyte complex. Alternatively, the labeling agent may be athird moiety, such as another antibody, that specifically binds to theantibody/protein complex.

In a preferred embodiment, the labeling agent is a second antibodybearing a label. Alternatively, the second antibody may lack a label,but it may, in turn, be bound by a labeled third antibody specific toantibodies of the species from which the second antibody is derived. Thesecond antibody can be modified with a detectable moiety, such asbiotin, to which a third labeled molecule can specifically bind, such asenzyme-labeled streptavidin.

Other proteins capable of specifically binding immunoglobulin constantregions, such as protein A or protein G, can also be used as the labelagents. These proteins are normal constituents of the cell walls ofstreptococcal bacteria. They exhibit a strong non-immunogenic reactivitywith immunoglobulin constant regions from a variety of species (see,generally, Kronval, et al. J. Immunol., 111:1401-1406 (1973); andAkerstrom, et al. J. Immunol., 135:2589-2542 (1985)).

Throughout the assays, incubation and/or washing steps may be requiredafter each combination of reagents. Incubation steps can vary from about5 seconds to several hours, preferably from about 5 minutes to about 24hours. The incubation time will depend upon the assay format, analyte,volume of solution, concentrations, and the like. Usually, the assayswill be carried out at ambient temperature, although they can beconducted over a range of temperatures, such as 10° C. to 40° C.

1. Non-Competitive Assay Formats

Immunoassays for detecting proteins of interest from tissue samples maybe either competitive or noncompetitive. Noncompetitive immunoassays areassays in which the amount of captured analyte (in this case theprotein) is directly measured. In one preferred “sandwich” assay, forexample, the capture agent (e.g., antibodies specific for a polypeptideencoded by a gene listed in Table 1-8) can be bound directly to a solidsubstrate where it is immobilized. These immobilized antibodies thencapture the polypeptide present in the test sample. The polypeptide thusimmobilized is then bound by a labeling agent, such as a second antibodybearing a label. Alternatively, the second antibody may lack a label,but it may, in turn, be bound by a labeled third antibody specific toantibodies of the species from which the second antibody is derived. Thesecond can be modified with a detectable moiety, such as biotin, towhich a third labeled molecule can specifically bind, such asenzyme-labeled streptavidin.

2. Competitive Assay Formats

In competitive assays, the amount of analyte (such as a polypeptideencoded by a gene listed in Table 1-8) present in the sample is measuredindirectly by measuring the amount of an added (exogenous) analytedisplaced (or competed away) from a capture agent (e.g., an antibodyspecific for the analyte) by the analyte present in the sample. In onecompetitive assay, a known amount of, in this case, the protein ofinterest is added to the sample and the sample is then contacted with acapture agent, in this case an antibody that specifically binds to apolypeptide of the invention. The amount of immunogen bound to theantibody is inversely proportional to the concentration of immunogenpresent in the sample. In a particularly preferred embodiment, theantibody is immobilized on a solid substrate. For example, the amount ofthe polypeptide bound to the antibody may be determined either bymeasuring the amount of subject protein present in a protein/antibodycomplex or, alternatively, by measuring the amount of remaininguncomplexed protein. The amount of protein may be detected by providinga labeled protein molecule.

Immunoassays in the competitive binding format can be used forcross-reactivity determinations. For example, a protein of interest canbe immobilized on a solid support. Proteins are added to the assay whichcompete with the binding of the antisera to the immobilized antigen. Theability of the above proteins to compete with the binding of theantisera to the immobilized protein is compared to that of the proteinof interest. The percent cross-reactivity for the above proteins iscalculated, using standard calculations. Those antisera with less than10% cross-reactivity with each of the proteins listed above are selectedand pooled. The cross-reacting antibodies are optionally removed fromthe pooled antisera by immunoabsorption with the considered proteins,e.g., distantly related homologs.

The immunoabsorbed and pooled antisera are then used in a competitivebinding immunoassay as described above to compare a second protein,thought to be perhaps a protein of the present invention, to theimmunogen protein. In order to make this comparison, the two proteinsare each assayed at a wide range of concentrations and the amount ofeach protein required to inhibit 50% of the binding of the antisera tothe immobilized protein is determined. If the amount of the secondprotein required is less than 10 times the amount of the proteinpartially encoded by a sequence herein that is required, then the secondprotein is said to specifically bind to an antibody generated to animmunogen consisting of the target protein.

3. Other Assay Formats

In a particularly preferred embodiment, western blot (immunoblot)analysis is used to detect and quantify the presence of a polypeptide ofthe invention in the sample. The technique generally comprisesseparating sample proteins by gel electrophoresis on the basis ofmolecular weight, transferring the separated proteins to a suitablesolid support (such as, e.g., a nitrocellulose filter, a nylon filter,or a derivatized nylon filter) and incubating the sample with theantibodies that specifically bind the protein of interest. For example,the antibodies specifically bind to a polypeptide of interest on thesolid support. These antibodies may be directly labeled or alternativelymay be subsequently detected using labeled antibodies (e.g., labeledsheep anti-mouse antibodies) that specifically bind to the antibodiesagainst the protein of interest.

Other assay formats include liposome immunoassays (LIA), which useliposomes designed to bind specific molecules (e.g., antibodies) andrelease encapsulated reagents or markers. The released chemicals arethen detected according to standard techniques (see, Monroe et al.(1986) Amer. Clin. Prod. Rev. 5:34-41).

4. Labels

The particular label or detectable group used in the assay is not acritical aspect of the invention, as long as it does not significantlyinterfere with the specific binding of the antibody used in the assay.The detectable group can be any material having a detectable physical orchemical property. Such detectable labels have been well developed inthe field of immunoassays and, in general, most labels useful in suchmethods can be applied to the present invention. Thus, a label is anycomposition detectable by spectroscopic, photochemical, biochemical,immunochemical, electrical, optical or chemical means. Useful labels inthe present invention include magnetic beads (e.g., Dynabeads™),fluorescent dyes (e.g., fluorescein isothiocyanate, Texas red,rhodamine, and the like), radiolabels (e.g., ³H, ¹²⁵I, ³⁵S, ¹⁴C, or³²P), enzymes (e.g., horse radish peroxidase, alkaline phosphatase andothers commonly used in an ELISA), and colorimetric labels such ascolloidal gold or colored glass or plastic (e.g., polystyrene,polypropylene, latex, etc.) beads.

The label may be coupled directly or indirectly to the desired componentof the assay according to methods well known in the art. As indicatedabove, a wide variety of labels may be used, with the choice of labeldepending on the sensitivity required, the ease of conjugation with thecompound, stability requirements, available instrumentation, anddisposal provisions.

Non-radioactive labels are often attached by indirect means. Themolecules can also be conjugated directly to signal generatingcompounds, e.g., by conjugation with an enzyme or fluorescent compound.A variety of enzymes and fluorescent compounds can be used with themethods of the present invention and are well-known to those of skill inthe art (for a review of various labeling or signal producing systemswhich may be used, see, e.g., U.S. Pat. No. 4,391,904).

Means of detecting labels are well known to those of skill in the art.Thus, for example, where the label is a radioactive label, means fordetection include a scintillation counter or photographic film as inautoradiography. Where the label is a fluorescent label, it may bedetected by exciting the fluorochrome with the appropriate wavelength oflight and detecting the resulting fluorescence. The fluorescence may bedetected visually, by means of photographic film, by the use ofelectronic detectors such as charge-coupled devices (CCDs) orphotomultipliers and the like. Similarly, enzymatic labels may bedetected by providing the appropriate substrates for the enzyme anddetecting the resulting reaction product. Finally simple calorimetriclabels may be detected directly by observing the color associated withthe label. Thus, in various dipstick assays, conjugated gold oftenappears pink, while various conjugated beads appear the color of thebead.

Some assay formats do not require the use of labeled components. Forinstance, agglutination assays can be used to detect the presence of thetarget antibodies. In this case, antigen-coated particles areagglutinated by samples comprising the target antibodies. In thisformat, none of the components need to be labeled and the presence ofthe target antibody is detected by simple visual inspection.

VI. Screening for Modulators of Polypeptides and Polynucleotides of theInvention

Modulators of polypeptides or polynucleotides of the invention, i.e.agonists or antagonists of their activity or modulators of polypeptideor polynucleotide expression, are useful for treating a number of humandiseases, including mood disorders or psychotic disorders.Administration of agonists, antagonists or other agents that modulateexpression of the polynucleotides or polypeptides of the invention canbe used to treat patients with mood disorders or psychotic disorders.

A. Screening Methods

A number of different screening protocols can be utilized to identifyagents that modulate the level of expression or activity of polypeptidesand polynucleotides of the invention in cells, particularly mammaliancells, and especially human cells. In general terms, the screeningmethods involve screening a plurality of agents to identify an agentthat modulates the polypeptide activity by binding to a polypeptide ofthe invention, modulating inhibitor binding to the polypeptide oractivating expression of the polypeptide or polynucleotide, for example.

1. Binding Assays

Preliminary screens can be conducted by screening for agents capable ofbinding to a polypeptide of the invention, as at least some of theagents so identified are likely modulators of polypeptide activity. Thebinding assays usually involve contacting a polypeptide of the inventionwith one or more test agents and allowing sufficient time for theprotein and test agents to form a binding complex. Any binding complexesformed can be detected using any of a number of established analyticaltechniques. Protein binding assays include, but are not limited to,methods that measure co-precipitation, co-migration on non-denaturingSDS-polyacrylamide gels, and co-migration on Western blots (see, e.g.,Bennet and Yamamura, (1985) “Neurotransmitter, Hormone or Drug ReceptorBinding Methods,” in Neurotransmitter Receptor Binding (Yamamura, H. I.,et al., eds.), pp. 61-89. The protein utilized in such assays can benaturally expressed, cloned or synthesized.

Binding assays are also useful, e.g., for identifying endogenousproteins that interact with a polypeptide of the invention. For example,antibodies, receptors or other molecules that bind a polypeptide of theinvention can be identified in binding assays.

2. Expression Assays

Certain screening methods involve screening for a compound that up ordown-regulates the expression of a polypeptide or polynucleotide of theinvention. Such methods generally involve conducting cell-based assaysin which test compounds are contacted with one or more cells expressinga polypeptide or polynucleotide of the invention and then detecting anincrease or decrease in expression (either transcript, translationproduct, or catalytic product). Some assays are performed withperipheral cells, or other cells, that express an endogenous polypeptideor polynucleotide of the invention.

Polypeptide or polynucleotide expression can be detected in a number ofdifferent ways. As described infra, the expression level of apolynucleotide of the invention in a cell can be determined by probingthe mRNA expressed in a cell with a probe that specifically hybridizeswith a transcript (or complementary nucleic acid derived therefrom) of apolynucleotide of the invention. Probing can be conducted by lysing thecells and conducting Northern blots or without lysing the cells using insitu-hybridization techniques. Alternatively, a polypeptide of theinvention can be detected using immunological methods in which a celllysate is probed with antibodies that specifically bind to a polypeptideof the invention.

Other cell-based assays are reporter assays conducted with cells that donot express a polypeptide or polynucleotide of the invention. Certain ofthese assays are conducted with a heterologous nucleic acid constructthat includes a promoter of a polynucleotide of the invention that isoperably linked to a reporter gene that encodes a detectable product. Anumber of different reporter genes can be utilized. Some reporters areinherently detectable. An example of such a reporter is greenfluorescent protein that emits fluorescence that can be detected with afluorescence detector. Other reporters generate a detectable product.Often such reporters are enzymes. Exemplary enzyme reporters include,but are not limited to, β-glucuronidase, chloramphenicol acetyltransferase (CAT); Alton and Vapnek (1979) Nature 282:864-869),luciferase, β-galactosidase, green fluorescent protein (GFP) andalkaline phosphatase (Toh, et al. (1980) Eur. J. Biochem. 182:231-238;and Hall et al. (1983) J. Mol. Appl. Gen. 2:101).

In these assays, cells harboring the reporter construct are contactedwith a test compound. A test compound that either activates the promoterby binding to it or triggers a cascade that produces a molecule thatactivates the promoter causes expression of the detectable reporter.Certain other reporter assays are conducted with cells that harbor aheterologous construct that includes a transcriptional control elementthat activates expression of a polynucleotide of the invention and areporter operably linked thereto. Here, too, an agent that binds to thetranscriptional control element to activate expression of the reporteror that triggers the formation of an agent that binds to thetranscriptional control element to activate reporter expression, can beidentified by the generation of signal associated with reporterexpression.

The level of expression or activity can be compared to a baseline value.As indicated above, the baseline value can be a value for a controlsample or a statistical value that is representative of expressionlevels for a control population (e.g., healthy individuals not having orat risk for mood disorders or psychotic disorders). Expression levelscan also be determined for cells that do not express a polynucleotide ofthe invention as a negative control. Such cells generally are otherwisesubstantially genetically the same as the test cells.

A variety of different types of cells can be utilized in the reporterassays. Cells that express an endogenous polypeptide or polynucleotideof the invention include, e.g., brain cells, including cells from thecerebellum, anterior cingulate cortex, or dorsolateral prefrontalcortex. Such brain regions are part of brain circuits or pathways thatare implicated in mood disorders. Cells that do not endogenously expresspolynucleotides of the invention can be prokaryotic, but are preferablyeukaryotic. The eukaryotic cells can be any of the cells typicallyutilized in generating cells that harbor recombinant nucleic acidconstructs. Exemplary eukaryotic cells include, but are not limited to,yeast, and various higher eukaryotic cells such as the COS, CHO and HeLacell lines, and stem cells.

Various controls can be conducted to ensure that an observed activity isauthentic including running parallel reactions with cells that lack thereporter construct or by not contacting a cell harboring the reporterconstruct with test compound. Compounds can also be further validated asdescribed below.

3. Catalytic Activity

Catalytic activity of polypeptides of the invention can be determined bymeasuring the production of enzymatic products or by measuring theconsumption of substrates. Activity refers to either the rate ofcatalysis or the ability to the polypeptide to bind (K_(m)) thesubstrate or release the catalytic product (K_(d)).

Analysis of the activity of polypeptides of the invention are performedaccording to general biochemical analyses. Such assays includecell-based assays as well as in vitro assays involving purified orpartially purified polypeptides or crude cell lysates. The assaysgenerally involve providing a known quantity of substrate andquantifying product as a function of time.

4. Validation

Agents that are initially identified by any of the foregoing screeningmethods can be further tested to validate the apparent activity.Preferably such studies are conducted with suitable animal models. Thebasic format of such methods involves administering a lead compoundidentified during an initial screen to an animal that serves as a modelfor humans and then determining if expression or activity of apolynucleotide or polypeptide of the invention is in fact upregulated.The animal models utilized in validation studies generally are mammalsof any kind. Specific examples of suitable animals include, but are notlimited to, primates, mice, and rats.

5. Animal Models

Animal models of mental disorders also find use in screening formodulators. In one embodiment, rat models of depression (both chronicand acute), in which the rats are subjected to stress, are used forscreening. In one embodiment, invertebrate models such as Drosophilamodels can be used, screening for modulators of Drosophila orthologs ofthe human genes disclosed herein. In another embodiment, transgenicanimal technology including gene knockout technology, for example as aresult of homologous recombination with an appropriate gene targetingvector, or gene overexpression, will result in the absence, decreased orincreased expression of a polynucleotide or polypeptide of theinvention. The same technology can also be applied to make knockoutcells. When desired, tissue-specific expression or knockout of apolynucleotide or polypeptide of the invention may be necessary.Transgenic animals generated by such methods find use as animal modelsof mental disorders and are useful in screening for modulators of mentaldisorders.

Knockout cells and transgenic mice can be made by insertion of a markergene or other heterologous gene into an endogenous gene site in themouse genome via homologous recombination. Such mice can also be made bysubstituting an endogenous polynucleotide of the invention with amutated version of the polynucleotide, or by mutating an endogenouspolynucleotide, e.g., by exposure to carcinogens.

For development of appropriate stem cells, a DNA construct is introducedinto the nuclei of embryonic stem cells. Cells containing the newlyengineered genetic lesion are injected into a host mouse embryo, whichis re-implanted into a recipient female. Some of these embryos developinto chimeric mice that possess germ cells partially derived from themutant cell line. Therefore, by breeding the chimeric mice it ispossible to obtain a new line of mice containing the introduced geneticlesion (see, e.g., Capecchi et al., Science 244:1288 (1989)). Chimerictargeted mice can be derived according to Hogan et al., Manipulating theMouse Embryo: A Laboratory Manual, Cold Spring Harbor Laboratory (1988)and Teratocarcinomas and Embryonic Stem Cells: A Practical Approach,Robertson, ed., IRL Press, Washington, D.C., (1987).

B. Modulators of Polypeptides or Polynucleotides of the Invention

The agents tested as modulators of the polypeptides or polynucleotidesof the invention can be any small chemical compound, or a biologicalentity, such as a protein, sugar, nucleic acid or lipid. Alternatively,modulators can be genetically altered versions of a polypeptide orpolynucleotide of the invention. Typically, test compounds will be smallchemical molecules and peptides. Essentially any chemical compound canbe used as a potential modulator or ligand in the assays of theinvention, although most often compounds that can be dissolved inaqueous or organic (especially DMSO-based) solutions are used. Theassays are designed to screen large chemical libraries by automating theassay steps and providing compounds from any convenient source toassays, which are typically run in parallel (e.g., in microtiter formatson microtiter plates in robotic assays). It will be appreciated thatthere are many suppliers of chemical compounds, including Sigma (St.Louis, Mo.), Aldrich (St. Louis, Mo.), Sigma-Aldrich (St. Louis, Mo.),Fluka Chemika-Biochemica Analytika (Buchs, Switzerland) and the like.Modulators also include agents designed to reduce the level of mRNA ofthe invention (e.g. antisense molecules, ribozymes, DNAzymes and thelike) or the level of translation from an mRNA.

In one preferred embodiment, high throughput screening methods involveproviding a combinatorial chemical or peptide library containing a largenumber of potential therapeutic compounds (potential modulator or ligandcompounds). Such “combinatorial chemical libraries” or “ligandlibraries” are then screened in one or more assays, as described herein,to identify those library members (particular chemical species orsubclasses) that display a desired characteristic activity. Thecompounds thus identified can serve as conventional “lead compounds” orcan themselves be used as potential or actual therapeutics.

A combinatorial chemical library is a collection of diverse chemicalcompounds generated by either chemical synthesis or biologicalsynthesis, by combining a number of chemical “building blocks” such asreagents. For example, a linear combinatorial chemical library such as apolypeptide library is formed by combining a set of chemical buildingblocks (amino acids) in every possible way for a given compound length(i.e., the number of amino acids in a polypeptide compound). Millions ofchemical compounds can be synthesized through such combinatorial mixingof chemical building blocks.

Preparation and screening of combinatorial chemical libraries is wellknown to those of skill in the art. Such combinatorial chemicallibraries include, but are not limited to, peptide libraries (see, e.g.,U.S. Pat. No. 5,010,175, Furka, Int. J. Pept. Prot. Res. 37:487-493(1991) and Houghton et al., Nature 354:84-88 (1991)). Other chemistriesfor generating chemical diversity libraries can also be used. Suchchemistries include, but are not limited to: peptoids (e.g., PCTPublication No. WO 91/19735), encoded peptides (e.g., PCT Publication WO93/20242), random bio-oligomers (e.g., PCT Publication No. WO 92/00091),benzodiazepines (e.g., U.S. Pat. No. 5,288,514), diversomers such ashydantoins, benzodiazepines and dipeptides (Hobbs et al., Proc. Nat.Acad. Sci. USA 90:6909-6913 (11993)), vinylogous polypeptides (Hagiharaet al., J. Amer. Chem. Soc. 114:6568 (1992)), nonpeptidalpeptidomimetics with glucose scaffolding (Hirschmann et al., J. Amer.Chem. Soc. 114:9217-9218 (1992)), analogous organic syntheses of smallcompound libraries (Chen et al., J. Amer. Chem. Soc. 116:2661 (1994)),oligocarbamates (Cho et al., Science 261:1303 (1993)), and/or peptidylphosphonates (Campbell et al., J. Org. Chem. 59:658 (1994)), nucleicacid libraries (see Ausubel, Berger and Sambrook, all supra), peptidenucleic acid libraries (see, e.g., U.S. Pat. No. 5,539,083), antibodylibraries (see, e.g., Vaughn et al., Nature Biotechnology, 14(3):309-314(1996) and PCT/US96/10287), carbohydrate libraries (see, e.g., Liang etal., Science, 274:1520-1522 (1996) and U.S. Pat. No. 5,593,853), smallorganic molecule libraries (see, e.g., benzodiazepines, Baum C&EN,January 18, page 33 (1993); isoprenoids, U.S. Pat. No. 5,569,588;thiazolidinones and metathiazanones, U.S. Pat. No. 5,549,974;pyrrolidines, U.S. Pat. Nos. 5,525,735 and 5,519,134; morpholinocompounds, U.S. Pat. Nos. 5,506,337; benzodiazepines, 5,288,514, and thelike).

Devices for the preparation of combinatorial libraries are commerciallyavailable (see, e.g., 357 MPS, 390 MPS, Advanced Chem Tech, LouisvilleKy.; Symphony, Rainin, Woburn, Mass.; 433A Applied Biosystems, FosterCity, Calif.; 9050 Plus, Millipore, Bedford, Mass.). In addition,numerous combinatorial libraries are themselves commercially available(see, e.g., ComGenex, Princeton, N.J.; Tripos, Inc., St. Louis, Mo.; 3DPharmaceuticals, Exton, Pa.; Martek Biosciences, Columbia, Md., etc.).

C. Solid State and Soluble High Throughput Assays

In the high throughput assays of the invention, it is possible to screenup to several thousand different modulators or ligands in a single day.In particular, each well of a microtiter plate can be used to run aseparate assay against a selected potential modulator, or, ifconcentration or incubation time effects are to be observed, every 5-10wells can test a single modulator. Thus, a single standard microtiterplate can assay about 100 (e.g., 96) modulators. If 1536 well plates areused, then a single plate can easily assay from about 100 to about 1500different compounds. It is possible to assay several different platesper day; assay screens for up to about 6,000-20,000 different compoundsare possible using the integrated systems of the invention. Morerecently, microfluidic approaches to reagent manipulation have beendeveloped.

The molecule of interest can be bound to the solid state component,directly or indirectly, via covalent or non-covalent linkage, e.g., viaa tag. The tag can be any of a variety of components. In general, amolecule that binds the tag (a tag binder) is fixed to a solid support,and the tagged molecule of interest is attached to the solid support byinteraction of the tag and the tag binder.

A number of tags and tag binders can be used, based upon known molecularinteractions well described in the literature. For example, where a taghas a natural binder, for example, biotin, protein A, or protein G, itcan be used in conjunction with appropriate tag binders (avidin,streptavidin, neutravidin, the Fc region of an immunoglobulin, etc.).Antibodies to molecules with natural binders such as biotin are alsowidely available and appropriate tag binders (see, SIGMA Immunochemicals1998 catalogue SIGMA, St. Louis Mo.).

Similarly, any haptenic or antigenic compound can be used in combinationwith an appropriate antibody to form a tag/tag binder pair. Thousands ofspecific antibodies are commercially available and many additionalantibodies are described in the literature. For example, in one commonconfiguration, the tag is a first antibody and the tag binder is asecond antibody which recognizes the first antibody. In addition toantibody-antigen interactions, receptor-ligand interactions are alsoappropriate as tag and tag-binder pairs, such as agonists andantagonists of cell membrane receptors (e.g., cell receptor-ligandinteractions such as transferrin, c-kit, viral receptor ligands,cytokine receptors, chemokine receptors, interleukin receptors,immunoglobulin receptors and antibodies, the cadherin family, theintegrin family, the selectin family, and the like; see, e.g., Pigott &Power, The Adhesion Molecule Facts Book I (1993)). Similarly, toxins andvenoms, viral epitopes, hormones (e.g., opiates, steroids, etc.),intracellular receptors (e.g., which mediate the effects of varioussmall ligands, including steroids, thyroid hormone, retinoids andvitamin D; peptides), drugs, lectins, sugars, nucleic acids (both linearand cyclic polymer configurations), oligosaccharides, proteins,phospholipids and antibodies can all interact with various cellreceptors.

Synthetic polymers, such as polyurethanes, polyesters, polycarbonates,polyureas, polyamides, polyethyleneimines, polyarylene sulfides,polysiloxanes, polyimides, and polyacetates can also form an appropriatetag or tag binder. Many other tag/tag binder pairs are also useful inassay systems described herein, as would be apparent to one of skillupon review of this disclosure.

Common linkers such as peptides, polyethers, and the like can also serveas tags, and include polypeptide sequences, such as poly-Gly sequencesof between about 5 and 200 amino acids. Such flexible linkers are knownto those of skill in the art. For example, poly(ethelyne glycol) linkersare available from Shearwater Polymers, Inc., Huntsville, Ala. Theselinkers optionally have amide linkages, sulfhydryl linkages, orheterofunctional linkages.

Tag binders are fixed to solid substrates using any of a variety ofmethods currently available. Solid substrates are commonly derivatizedor functionalized by exposing all or a portion of the substrate to achemical reagent which fixes a chemical group to the surface which isreactive with a portion of the tag binder. For example, groups which aresuitable for attachment to a longer chain portion would include amines,hydroxyl, thiol, and carboxyl groups. Aminoalkylsilanes andhydroxyalkylsilanes can be used to functionalize a variety of surfaces,such as glass surfaces. The construction of such solid phase biopolymerarrays is well described in the literature (see, e.g., Merrifield, J.Am. Chem. Soc. 85:2149-2154 (1963) (describing solid phase synthesis of,e.g., peptides); Geysen et al., J. Immun. Meth. 102:259-274 (1987)(describing synthesis of solid phase components on pins); Frank andDoring, Tetrahedron 44:60316040 (1988) (describing synthesis of variouspeptide sequences on cellulose disks); Fodor et al., Science,251:767-777 (1991); Sheldon et al., Clinical Chemistry 39(4):718-719(1993); and Kozal et al., Nature Medicine 2(7):753759 (1996) (alldescribing arrays of biopolymers fixed to solid substrates).Non-chemical approaches for fixing tag binders to substrates includeother common methods, such as heat, cross-linking by UV radiation, andthe like.

The invention provides in vitro assays for identifying, in a highthroughput format, compounds that can modulate the expression oractivity of the polynucleotides or polypeptides of the invention. In apreferred embodiment, the methods of the invention include such acontrol reaction. For each of the assay formats described, “nomodulator” control reactions that do not include a modulator provide abackground level of binding activity.

In some assays it will be desirable to have positive controls to ensurethat the components of the assays are working properly. At least twotypes of positive controls are appropriate. First, a known activator ofa polynucleotide or polypeptide of the invention can be incubated withone sample of the assay, and the resulting increase in signal resultingfrom an increased expression level or activity of polynucleotide orpolypeptide determined according to the methods herein. Second, a knowninhibitor of a polynucleotide or polypeptide of the invention can beadded, and the resulting decrease in signal for the expression oractivity can be similarly detected.

D. Computer-Based Assays

Yet another assay for compounds that modulate the activity of apolypeptide or polynucleotide of the invention involves computerassisted drug design, in which a computer system is used to generate athree-dimensional structure of the polypeptide or polynucleotide basedon the structural information encoded by its amino acid or nucleotidesequence. The input sequence interacts directly and actively with apre-established algorithm in a computer program to yield secondary,tertiary, and quaternary structural models of the molecule. Similaranalyses can be performed on potential receptors or binding partners ofthe polypeptides or polynucleotides of the invention. The models of theprotein or nucleotide structure are then examined to identify regions ofthe structure that have the ability to bind, e.g., a polypeptide orpolynucleotide of the invention. These regions are then used to identifypolypeptides that bind to a polypeptide or polynucleotide of theinvention.

The three-dimensional structural model of a protein is generated byentering protein amino acid sequences of at least 10 amino acid residuesor corresponding nucleic acid sequences encoding a potential receptorinto the computer system. The amino acid sequences encoded by thenucleic acid sequences provided herein represent the primary sequencesor subsequences of the proteins, which encode the structural informationof the proteins. At least 10 residues of an amino acid sequence (or anucleotide sequence encoding 10 amino acids) are entered into thecomputer system from computer keyboards, computer readable substratesthat include, but are not limited to, electronic storage media (e.g.,magnetic diskettes, tapes, cartridges, and chips), optical media (e.g.,CD ROM), information distributed by internet sites, and by RAM. Thethree-dimensional structural model of the protein is then generated bythe interaction of the amino acid sequence and the computer system,using software known to those of skill in the art.

The amino acid sequence represents a primary structure that encodes theinformation necessary to form the secondary, tertiary, and quaternarystructure of the protein of interest. The software looks at certainparameters encoded by the primary sequence to generate the structuralmodel. These parameters are referred to as “energy terms,” and primarilyinclude electrostatic potentials, hydrophobic potentials, solventaccessible surfaces, and hydrogen bonding. Secondary energy termsinclude van der Waals potentials. Biological molecules form thestructures that minimize the energy terms in a cumulative fashion. Thecomputer program is therefore using these terms encoded by the primarystructure or amino acid sequence to create the secondary structuralmodel.

The tertiary structure of the protein encoded by the secondary structureis then formed on the basis of the energy terms of the secondarystructure. The user at this point can enter additional variables such aswhether the protein is membrane bound or soluble, its location in thebody, and its cellular location, e.g., cytoplasmic, surface, or nuclear.These variables along with the energy terms of the secondary structureare used to form the model of the tertiary structure. In modeling thetertiary structure, the computer program matches hydrophobic faces ofsecondary structure with like, and hydrophilic faces of secondarystructure with like.

Once the structure has been generated, potential ligand binding regionsare identified by the computer system. Three-dimensional structures forpotential ligands are generated by entering amino acid or nucleotidesequences or chemical formulas of compounds, as described above. Thethree-dimensional structure of the potential ligand is then compared tothat of a polypeptide or polynucleotide of the invention to identifybinding sites of the polypeptide or polynucleotide of the invention.Binding affinity between the protein and ligands is determined usingenergy terms to determine which ligands have an enhanced probability ofbinding to the protein.

Computer systems are also used to screen for mutations, polymorphicvariants, alleles and interspecies homologs of genes encoding apolypeptide or polynucleotide of the invention. Such mutations can beassociated with disease states or genetic traits and can be used fordiagnosis. As described above, GeneChip™ and related technology can alsobe used to screen for mutations, polymorphic variants, alleles andinterspecies homologs. Once the variants are identified, diagnosticassays can be used to identify patients having such mutated genes.Identification of the mutated a polypeptide or polynucleotide of theinvention involves receiving input of a first amino acid sequence of apolypeptide of the invention (or of a first nucleic acid sequenceencoding a polypeptide of the invention), e.g., any amino acid sequencehaving at least 60%, optionally at least 70% or 85%, identity with theamino acid sequence of interest, or conservatively modified versionsthereof. The sequence is entered into the computer system as describedabove. The first nucleic acid or amino acid sequence is then compared toa second nucleic acid or amino acid sequence that has substantialidentity to the first sequence. The second sequence is entered into thecomputer system in the manner described above. Once the first and secondsequences are compared, nucleotide or amino acid differences between thesequences are identified. Such sequences can represent allelicdifferences in various polynucleotides, including SNPs and/orhaplotypes, of the invention, and mutations associated with diseasestates and genetic traits.

VII. Compositions, Kits and Integrated Systems

The invention provides compositions, kits and integrated systems forpracticing the assays described herein using polypeptides orpolynucleotides of the invention, antibodies specific for polypeptidesor polynucleotides of the invention, etc.

The invention provides assay compositions for use in solid phase assays;such compositions can include, for example, one or more polynucleotidesor polypeptides of the invention immobilized on a solid support, and alabeling reagent. In each case, the assay compositions can also includeadditional reagents that are desirable for hybridization. Modulators ofexpression or activity of polynucleotides or polypeptides of theinvention can also be included in the assay compositions.

The invention also provides kits for carrying out the therapeutic anddiagnostic assays of the invention. The kits typically include a probethat comprises an antibody that specifically binds to polypeptides orpolynucleotides of the invention, and a label for detecting the presenceof the probe. The kits may include several polynucleotide sequencesencoding polypeptides of the invention. Kits can include any of thecompositions noted above, and optionally further include additionalcomponents such as instructions to practice a high-throughput method ofassaying for an effect on expression of the genes encoding thepolypeptides of the invention, or on activity of the polypeptides of theinvention, one or more containers or compartments (e.g., to hold theprobe, labels, or the like), a control modulator of the expression oractivity of polypeptides of the invention, a robotic armature for mixingkit components or the like.

The invention also provides integrated systems for high-throughputscreening of potential modulators for an effect on the expression oractivity of the polypeptides of the invention. The systems typicallyinclude a robotic armature which transfers fluid from a source to adestination, a controller which controls the robotic armature, a labeldetector, a data storage unit which records label detection, and anassay component such as a microtiter dish comprising a well having areaction mixture or a substrate comprising a fixed nucleic acid orimmobilization moiety.

A number of robotic fluid transfer systems are available, or can easilybe made from existing components. For example, a Zymate XP (ZymarkCorporation; Hopkinton, Mass.) automated robot using a Microlab 2200(Hamilton; Reno, Nev.) pipetting station can be used to transferparallel samples to 96 well microtiter plates to set up several parallelsimultaneous STAT binding assays.

Optical images viewed (and, optionally, recorded) by a camera or otherrecording device (e.g., a photodiode and data storage device) areoptionally further processed in any of the embodiments herein, e.g., bydigitizing the image and storing and analyzing the image on a computer.A variety of commercially available peripheral equipment and software isavailable for digitizing, storing and analyzing a digitized video ordigitized optical image, e.g., using PC, MACINTOSH®, or UNIX® based(e.g., SUN® work station) computers.

One conventional system carries light from the specimen field to acooled charge-coupled device (CCD) camera, in common use in the art. ACCD camera includes an array of picture elements (pixels). The lightfrom the specimen is imaged on the CCD. Particular pixels correspondingto regions of the specimen (e.g., individual hybridization sites on anarray of biological polymers) are sampled to obtain light intensityreadings for each position. Multiple pixels are processed in parallel toincrease speed. The apparatus and methods of the invention are easilyused for viewing any sample, e.g., by fluorescent or dark fieldmicroscopic techniques. Lasar based systems can also be used.

VIII. Administration and Pharmaceutical Compositions

Modulators of the polynucleotides or polypeptides of the invention(e.g., antagonists or agonists) can be administered directly to amammalian subject for modulation of activity of those molecules in vivo.Administration is by any of the routes normally used for introducing amodulator compound into ultimate contact with the tissue to be treatedand is well known to those of skill in the art. Although more than oneroute can be used to administer a particular composition, a particularroute can often provide a more immediate and more effective reactionthan another route.

Diseases that can be treated include the following, which include thecorresponding reference number from Morrison, DSM-IV Made Easy, 1995:Schizophrenia, Catatonic, Subchronic, (295.21); Schizophrenia,Catatonic, Chronic (295.22); Schizophrenia, Catatonic, Subchronic withAcute Exacerbation (295.23); Schizophrenia, Catatonic, Chronic withAcute Exacerbation (295.24); Schizophrenia, Catatonic, in Remission(295.55); Schizophrenia, Catatonic, Unspecified (295.20); Schizophrenia,Disorganized, Subchronic (295.11); Schizophrenia, Disorganized, Chronic(295.12); Schizophrenia, Disorganized, Subchronic with AcuteExacerbation (295.13); Schizophrenia, Disorganized, Chronic with AcuteExacerbation (295.14); Schizophrenia, Disorganized, in Remission(295.15); Schizophrenia, Disorganized, Unspecified (295.10);Schizophrenia, Paranoid, Subchronic (295.31); Schizophrenia, Paranoid,Chronic (295.32); Schizophrenia, Paranoid, Subchronic with AcuteExacerbation (295.33); Schizophrenia, Paranoid, Chronic with AcuteExacerbation (295.34); Schizophrenia, Paranoid, in Remission (295.35);Schizophrenia, Paranoid, Unspecified (295.30); Schizophrenia,Undifferentiated, Subchronic (295.91); Schizophrenia, Undifferentiated,Chronic (295.92); Schizophrenia, Undifferentiated, Subchronic with AcuteExacerbation (295.93); Schizophrenia, Undifferentiated, Chronic withAcute Exacerbation (295.94); Schizophrenia, Undifferentiated, inRemission (295.95); Schizophrenia, Undifferentiated, Unspecified(295.90); Schizophrenia, Residual, Subchronic (295.61); Schizophrenia,Residual, Chronic (295.62); Schizophrenia, Residual, Subchronic withAcute Exacerbation (295.63); Schizophrenia, Residual, Chronic with AcuteExacerbation (295.94); Schizophrenia, Residual, in Remission (295.65);Schizophrenia, Residual, Unspecified (295.60); Delusional (Paranoid)Disorder (297.10); Brief Reactive Psychosis (298.80); SchizophreniformDisorder (295.40); Schizoaffective Disorder (295.70); Induced PsychoticDisorder (297.30); Psychotic Disorder NOS (Atypical Psychosis) (298.90);Personality Disorders, Paranoid (301.00); Personality Disorders,Schizoid (301.20); Personality Disorders, Schizotypal (301.22);Personality Disorders, Antisocial (301.70); Personality Disorders,Borderline (301.83) and bipolar disorders, maniac, hypomaniac, dysthymicor cyclothymic disorders, substance-induced mood disorders, majordepression, psychotic disorders, including paranoid psychosis, catatonicpsychosis, delusional psychosis, having schizoaffective disorder, andsubstance-induced psychotic disorder.

In some embodiments, modulators of polynucleotides or polypeptides ofthe invention can be combined with other drugs useful for treatingmental disorders including useful for treating mood disorders, e.g.,schizophrenia, bipolar disorders, or major depression. In some preferredembodiments, pharmaceutical compositions of the invention comprise amodulator of a polypeptide of polynucleotide of the invention combinedwith at least one of the compounds useful for treating schizophrenia,bipolar disorder, or major depression, e.g., such as those described inU.S. Pat. Nos. 6,297,262; 6,284,760; 6,284,771; 6,232,326; 6,187,752;6,117,890; 6,239,162 or 6,166,008.

The pharmaceutical compositions of the invention may comprise apharmaceutically acceptable carrier. Pharmaceutically acceptablecarriers are determined in part by the particular composition beingadministered, as well as by the particular method used to administer thecomposition. Accordingly, there is a wide variety of suitableformulations of pharmaceutical compositions of the present invention(see, e.g., Remington's Pharmaceutical Sciences, 17^(th) ed. 1985)).

The modulators (e.g., agonists or antagonists) of the expression oractivity of the a polypeptide or polynucleotide of the invention, aloneor in combination with other suitable components, can be made intoaerosol formulations (i.e., they can be “nebulized”) to be administeredvia inhalation or in compositions useful for injection. Aerosolformulations can be placed into pressurized acceptable propellants, suchas dichlorodifluoromethane, propane, nitrogen, and the like.

Formulations suitable for administration include aqueous and non-aqueoussolutions, isotonic sterile solutions, which can contain antioxidants,buffers, bacteriostats, and solutes that render the formulationisotonic, and aqueous and non-aqueous sterile suspensions that caninclude suspending agents, solubilizers, thickening agents, stabilizers,and preservatives. In the practice of this invention, can beadministered or example, orally, nasally, topically, intravenously,intraperitoneally, or intrathecally. The formulations of compounds canbe presented in unit-dose or multi-dose sealed containers, such asampoules and vials. Solutions and suspensions can be prepared fromsterile powders, granules, and tablets of the kind previously described.The modulators can also be administered as part of a prepared food ordrug.

The dose administered to a patient, in the context of the presentinvention should be sufficient to effect a beneficial response in thesubject over time. The optimal dose level for any patient will depend ona variety of factors including the efficacy of the specific modulatoremployed, the age, body weight, physical activity, and diet of thepatient, on a possible combination with other drugs, and on the severityof the mental disorder. The size of the dose also will be determined bythe existence, nature, and extent of any adverse side effects thataccompany the administration of a particular compound or vector in aparticular subject.

In determining the effective amount of the modulator to be administereda physician may evaluate circulating plasma levels of the modulator,modulator toxicity, and the production of anti-modulator antibodies. Ingeneral, the dose equivalent of a modulator is from about 1 ng/kg to 10mg/kg for a typical subject.

For administration, modulators of the present invention can beadministered at a rate determined by the LD-50 of the modulator, and theside effects of the modulator at various concentrations, as applied tothe mass and overall health of the subject. Administration can beaccomplished via single or divided doses.

IX. Gene Therapy Applications

A variety of human diseases can be treated by therapeutic approachesthat involve stably introducing a gene into a human cell such that thegene is transcribed and the gene product is produced in the cell.Diseases amenable to treatment by this approach include inheriteddiseases, including those in which the defect is in a single or multiplegenes. Gene therapy is also useful for treatment of acquired diseasesand other conditions. For discussions on the application of gene therapytowards the treatment of genetic as well as acquired diseases, see,Miller, Nature 357:455-460 (1992); and Mulligan, Science 260:926-932(1993).

In the context of the present invention, gene therapy can be used fortreating a variety of disorders and/or diseases in which thepolynucleotides and polypeptides of the invention has been implicated.For example, compounds, including polynucleotides, can be identified bythe methods of the present invention as effective in treating a mentaldisorder. Introduction by gene therapy of these polynucleotides can thenbe used to treat, e.g., mental disorders including mood disorders andpsychotic disorders.

A. Vectors for Gene Delivery

For delivery to a cell or organism, the polynucleotides of the inventioncan be incorporated into a vector. Examples of vectors used for suchpurposes include expression plasmids capable of directing the expressionof the nucleic acids in the target cell. In other instances, the vectoris a viral vector system wherein the nucleic acids are incorporated intoa viral genome that is capable of transfecting the target cell. In apreferred embodiment, the polynucleotides can be operably linked toexpression and control sequences that can direct expression of the genein the desired target host cells. Thus, one can achieve expression ofthe nucleic acid under appropriate conditions in the target cell.

B. Gene Delivery Systems

Viral vector systems useful in the expression of the nucleic acidsinclude, for example, naturally occurring or recombinant viral vectorsystems. Depending upon the particular application, suitable viralvectors include replication competent, replication deficient, andconditionally replicating viral vectors. For example, viral vectors canbe derived from the genome of human or bovine adenoviruses, vacciniavirus, herpes virus, adeno-associated virus, minute virus of mice (MVM),HIV, sindbis virus, and retroviruses (including but not limited to Roussarcoma virus), and MoMLV. Typically, the genes of interest are insertedinto such vectors to allow packaging of the gene construct, typicallywith accompanying viral DNA, followed by infection of a sensitive hostcell and expression of the gene of interest.

As used herein, “gene delivery system” refers to any means for thedelivery of a nucleic acid of the invention to a target cell. In someembodiments of the invention, nucleic acids are conjugated to a cellreceptor ligand for facilitated uptake (e.g., invagination of coatedpits and internalization of the endosome) through an appropriate linkingmoiety, such as a DNA linking moiety (Wu et al., J. Biol. Chem.263:14621-14624 (1988); WO 92/06180). For example, nucleic acids can belinked through a polylysine moiety to asialo-oromucocid, which is aligand for the asialoglycoprotein receptor of hepatocytes.

Similarly, viral envelopes used for packaging gene constructs thatinclude the nucleic acids of the invention can be modified by theaddition of receptor ligands or antibodies specific for a receptor topermit receptor-mediated endocytosis into specific cells (see, e.g., WO93/20221, WO 93/14188, and WO 94/06923). In some embodiments of theinvention, the DNA constructs of the invention are linked to viralproteins, such as adenovirus particles, to facilitate endocytosis(Curiel et al., Proc. Natl. Acad. Sci. U.S.A. 88:8850-8854 (1991)). Inother embodiments, molecular conjugates of the instant invention caninclude microtubule inhibitors (WO/9406922), synthetic peptidesmimicking influenza virus hemagglutinin (Plank et al., J. Biol. Chem.269:12918-12924 (1994)), and nuclear localization signals such as SV40 Tantigen (WO93/19768).

Retroviral vectors are also useful for introducing the nucleic acids ofthe invention into target cells or organisms. Retroviral vectors areproduced by genetically manipulating retroviruses. The viral genome ofretroviruses is RNA. Upon infection, this genomic RNA is reversetranscribed into a DNA copy which is integrated into the chromosomal DNAof transduced cells with a high degree of stability and efficiency. Theintegrated DNA copy is referred to as a provirus and is inherited bydaughter cells as is any other gene. The wild type retroviral genome andthe proviral DNA have three genes: the gag, the pol and the env genes,which are flanked by two long terminal repeat (LTR) sequences. The gaggene encodes the internal structural (nucleocapsid) proteins; the polgene encodes the RNA directed DNA polymerase (reverse transcriptase);and the env gene encodes viral envelope glycoproteins. The 5′ and 3′LTRs serve to promote transcription and polyadenylation of virion RNAs.Adjacent to the 5′ LTR are sequences necessary for reverse transcriptionof the genome (the tRNA primer binding site) and for efficientencapsulation of viral RNA into particles (the Psi site) (see, Mulligan,In: Experimental Manipulation of Gene Expression, Inouye (ed), 155-173(1983); Mann et al., Cell 33:153-159 (1983); Cone and Mulligan,Proceedings of the National Academy of Sciences, U.S.A., 81:6349-6353(1984)).

The design of retroviral vectors is well known to those of ordinaryskill in the art. In brief, if the sequences necessary for encapsidation(or packaging of retroviral RNA into infectious virions) are missingfrom the viral genome, the result is a cis-acting defect which preventsencapsidation of genomic RNA. However, the resulting mutant is stillcapable of directing the synthesis of all virion proteins. Retroviralgenomes from which these sequences have been deleted, as well as celllines containing the mutant genome stably integrated into the chromosomeare well known in the art and are used to construct retroviral vectors.Preparation of retroviral vectors and their uses are described in manypublications including, e.g., European Patent Application EPA 0 178 220;U.S. Pat. No. 4,405,712, Gilboa Biotechniques 4:504-512 (1986); Mann etal., Cell 33:153-159 (1983); Cone and Mulligan Proc. Natl. Acad. Sci.USA 81:6349-6353 (1984); Eglitis et al. Biotechniques 6:608-614 (1988);Miller et al. Biotechniques 7:981-990 (1989); Miller (1992) sipra;Mulligan (1993), supra; and WO 92/07943.

The retroviral vector particles are prepared by recombinantly insertingthe desired nucleotide sequence into a retrovirus vector and packagingthe vector with retroviral capsid proteins by use of a packaging cellline. The resultant retroviral vector particle is incapable ofreplication in the host cell but is capable of integrating into the hostcell genome as a proviral sequence containing the desired nucleotidesequence. As a result, the patient is capable of producing, for example,a polypeptide or polynucleotide of the invention and thus restore thecells to a normal phenotype.

Packaging cell lines that are used to prepare the retroviral vectorparticles are typically recombinant mammalian tissue culture cell linesthat produce the necessary viral structural proteins required forpackaging, but which are incapable of producing infectious virions. Thedefective retroviral vectors that are used, on the other hand, lackthese structural genes but encode the remaining proteins necessary forpackaging. To prepare a packaging cell line, one can construct aninfectious clone of a desired retrovirus in which the packaging site hasbeen deleted. Cells comprising this construct will express allstructural viral proteins, but the introduced DNA will be incapable ofbeing packaged. Alternatively, packaging cell lines can be produced bytransforming a cell line with one or more expression plasmids encodingthe appropriate core and envelope proteins. In these cells, the gag,pol, and env genes can be derived from the same or differentretroviruses.

A number of packaging cell lines suitable for the present invention arealso available in the prior art. Examples of these cell lines includeCrip, GPE86, PA317 and PG13 (see Miller et al., J. Virol. 65:2220-2224(1991)). Examples of other packaging cell lines are described in Coneand Mulligan Proceedings of the National Academy of Sciences, USA,81:6349-6353 (1984); Danos and Mulligan Proceedings of the NationalAcademy of Sciences, USA, 85:6460-6464 (1988); Eglitis et al. (1988),supra; and Miller (1990), supra.

Packaging cell lines capable of producing retroviral vector particleswith chimeric envelope proteins may be used. Alternatively, amphotropicor xenotropic envelope proteins, such as those produced by PA317 and GPXpackaging cell lines may be used to package the retroviral vectors.

In some embodiments of the invention, an antisense polynucleotide isadministered which hybridizes to a gene encoding a polypeptide of theinvention. The antisense polypeptide can be provided as an antisenseoligonucleotide (see, e.g., Murayama et al., Antisense Nucleic Acid DrugDev. 7:109-114 (1997)). Genes encoding an antisense nucleic acid canalso be provided; such genes can be introduced into cells by methodsknown to those of skill in the art. For example, one can introduce anantisense nucleotide sequence in a viral vector, such as, for example,in hepatitis B virus (see, e.g., Ji et al., J. Viral Hepat. 4:167-173(1997)), in adeno-associated virus (see, e.g., Xiao et al., Brain Res.756:76-83 (1997)), or in other systems including, but not limited, to anHVJ (Sendai virus)-liposome gene delivery system (see, e.g., Kaneda etal., Ann. NY Acad. Sci. 811:299-308 (1997)), a “peptide vector” (see,e.g., Vidal et al., CR Acad. Sci. III 32:279-287 (1997)), as a gene inan episomal or plasmid vector (see, e.g., Cooper et al., Proc. Natl.Acad. Sci. U.S.A. 94:6450-6455 (1997), Yew et al. Hum Gene Ther.8:575-584 (1997)), as a gene in a peptide-DNA aggregate (see, e.g.,Niidome et al., J. Biol. Chem. 272:15307-15312 (1997)), as “naked DNA”(see, e.g., U.S. Pat. Nos. 5,580,859 and 5,589,466), in lipidic vectorsystems (see, e.g., Lee et al., Crit. Rev Ther Drug Carrier Syst.14:173-206 (1997)), polymer coated liposomes (U.S. Pat. Nos. 5,213,804and 5,013,556), cationic liposomes (Epand et al., U.S. Pat. Nos.5,283,185; 5,578,475; 5,279,833; and 5,334,761), gas filled microspheres(U.S. Pat. No. 5,542,935), ligand-targeted encapsulated macromolecules(U.S. Pat. Nos. 5,108,921; 5,521,291; 5,554,386; and 5,166,320). Inanother embodiment, conditional expression systems, such as thosetypified by the tet-regulated systems and the RU-486 system, can be used(see, e.g., Gossen & Bujard, PNAS 89:5547 (1992); Oligino et al., GeneTher. 5:491-496 (1998); Wang et al., Gene Ther. 4:432-441 (1997);Neering et al., Blood 88:1147-1155 (1996); and Rendahl et al., Nat.Biotechnol. 16:757-761 (1998)). These systems impart small moleculecontrol on the expression of the target gene(s) of interest.

C. Pharmaceutical Formulations

When used for pharmaceutical purposes, the vectors used for gene therapyare formulated in a suitable buffer, which can be any pharmaceuticallyacceptable buffer, such as phosphate buffered saline or sodiumphosphate/sodium sulfate, Tris buffer, glycine buffer, sterile water,and other buffers known to the ordinarily skilled artisan such as thosedescribed by Good et al. Biochemistiy 5:467 (1966).

The compositions can additionally include a stabilizer, enhancer, orother pharmaceutically acceptable carriers or vehicles. Apharmaceutically acceptable carrier can contain a physiologicallyacceptable compound that acts, for example, to stabilize the nucleicacids of the invention and any associated vector. A physiologicallyacceptable compound can include, for example, carbohydrates, such asglucose, sucrose or dextrans; antioxidants, such as ascorbic acid orglutathione; chelating agents; low molecular weight proteins or otherstabilizers or excipients. Other physiologically acceptable compoundsinclude wetting agents, emulsifying agents, dispersing agents, orpreservatives, which are particularly useful for preventing the growthor action of microorganisms. Various preservatives are well known andinclude, for example, phenol and ascorbic acid. Examples of carriers,stabilizers, or adjuvants can be found in Remington's PharmaceuticalSciences, Mack Publishing Company, Philadelphia, Pa., 17th ed. (1985).

D. Administration of Formulations

The formulations of the invention can be delivered to any tissue ororgan using any delivery method known to the ordinarily skilled artisan.In some embodiments of the invention, the nucleic acids of the inventionare formulated in mucosal, topical, and/or buccal formulations,particularly mucoadhesive gel and topical gel formulations. Exemplarypermeation enhancing compositions, polymer matrices, and mucoadhesivegel preparations for transdermal delivery are disclosed in U.S. Pat. No.5,346,701.

E. Methods of Treatment

The gene therapy formulations of the invention are typicallyadministered to a cell. The cell can be provided as part of a tissue,such as an epithelial membrane, or as an isolated cell, such as intissue culture. The cell can be provided in vivo, ex vivo, or in vitro.

The formulations can be introduced into the tissue of interest in vivoor ex vivo by a variety of methods. In some embodiments of theinvention, the nucleic acids of the invention are introduced into cellsby such methods as microinjection, calcium phosphate precipitation,liposome fusion, or biolistics. In further embodiments, the nucleicacids are taken up directly by the tissue of interest.

In some embodiments of the invention, the nucleic acids of the inventionare administered ex vivo to cells or tissues explanted from a patient,then returned to the patient. Examples of ex vivo administration oftherapeutic gene constructs include Nolta et al., Proc Natl. Acad. Sci.USA 93(6):2414-9 (1996); Koc et al., Seminars in Oncology 23 (1):46-65(1996); Raper et al., Annals of Surgery 223(2):116-26 (1996); Dalesandroet al., J. Thorac. Cardi. Surg., 11(2):416-22 (1996); and Makarov etal., Proc. Natl. Acad. Sci. USA 93(1):402-6 (1996).

X. Diagnosis of Mood Disorders and Psychotic Disorders

The present invention also provides methods of diagnosing mood disorders(such as major depression or bipolar disorder), psychotic disorders(such as schizophrenia) Diagnosis involves determining the level of apolypeptide or polynucleotide of the invention in a patient and thencomparing the level to a baseline or range. Typically, the baselinevalue is representative of a polypeptide or polynucleotide of theinvention in a healthy person not suffering from a mood disorder orpsychotic disorder or under the effects of medication or other drugs.Variation of levels of a polypeptide or polynucleotide of the inventionfrom the baseline range (either up or down) indicates that the patienthas a mood disorder or psychotic disorder or at risk of developing atleast some aspects of a mood disorder or psychotic disorder. In someembodiments, the level of a polypeptide or polynucleotide of theinvention are measured by taking a blood, urine or tissue sample from apatient and measuring the amount of a polypeptide or polynucleotide ofthe invention in the sample using any number of detection methods, suchas those discussed herein, e.g., SNPs or haplotypes associated with thisgenes.

In some embodiments, the level of the enzymatic product of a polypeptideor polynucleotide of the invention is measured and compared to abaseline value of a healthy person or persons. Modulated levels of theproduct compared to the baseline indicates that the patient has a mooddisorder or psychotic disorder or is at risk of developing at least someaspects of a mood disorder or psychotic disorder. Patient samples, forexample, can be blood, saliva, CSF, urine or tissue samples.

It is understood that the examples and embodiments described herein arefor illustrative purposes only and that various modifications or changesin light thereof will be suggested to persons skilled in the art and areto be included within the spirit and purview of this application andscope of the appended claims.

EXAMPLES Example 1 Identification of Genes Dysregulated in MoodDisorders

A total of twenty mood disorder brains (9 bipolar and 11 majordepression disorder patients) with twenty control brains were used inthis study. Each brain pair (case and control) was matched on the basisof gender, age, and postmortem interval. Three brain regions,dorsolateral prefrontal cortex (DLPFC), anterior cingulate cortex (AnCg)and the cerebellum (CB) were extracted for RNA and subjected tomicroarray analysis using Affymetrix oligonucleotide GeneChips™. EachRNA sample was subjected to two independent analyses. The results wereanalyzed using multiple statistical tools and algorithms with variousstringencies. Real time PCR analysis was used to confirm differentialgene expression for selected genes. The genes identified using thisstudy are listed in Tables 1, 2, and 3. Furthermore, biochemicalpathways associated with the differentially expressed genes wereidentified (see FIGS. 1-5).

The two cortical regions DLPFC and AnCg had similar gene expressionprofiles in controls but differed significantly in MDD and BP,demonstrating distinct gene expression profiles. BP subject showed morechanges in AnCg compared to DLPFC whereas MDD show less profound changesin both cortical regions but had greater effects in the DLPFC than inthe AnCg. For BP, several candidate genes were located in chromosomalregion 15q11-13, which is associated with the Prader-Willi syndrome (seeFIGS. 6-8).

Example 2 Identification of Additional Genes Dysregulated in MoodDisorders

The RNA from three brain regions, dorsolateral prefrontal cortex(DLPFC), anterior cingulate cortex (AnCg) and the cerebellum (CB) fromdeceased patients diagnosed with bipolar disease or major depression,and matched controls were extracted and subjected to microarray analysisusing Affymetrix oligonucleotide GeneChips™. The patient's particularconditions in their terminal phase (agonal factors, e.g., seizure, coma,hypoxia, dehydration, and pyrexia) and the conditions of the braintissue after death (postmortem factors, e.g., postmortem interval, andfreezer interval) are two major influences on RNA preservation inpostmortem brain tissue. Brain pH has been evaluated as an indicator foragonal status, and as an indicator of RNA preservation. The effect ofagonal factors and pH were taken into account for quality control of theRNA. Two RNA samples were subjected to independent analyses. The resultswere analyzed using multiple statistical tools and algorithms withvarious stringencies. The 967 genes identified using this study arelisted in Table 4. Real time PCR analysis was used to confirmdifferential gene expression for selected genes. Real time PCRconfirmation of differential gene expression for selected genes islisted in Table 5.

Furthermore, biochemical pathways associated with the differentiallyexpressed genes were identified. In particular, cortical areas in BPpatients showed activation of several pathways, including the proteasomepathway, the oxidative phosphorylation pathway, the ATP synthesispathway, and chaperones (i.e., heat shock proteins). In addition,signaling pathways dysregulated in BP include, e.g., G-coupled proteinreceptors, the phosphatidylinositol pathway, the cAMP pathway, themitogen activated protein kinase pathway, cytoskeletal systems, and thecortical GABA and glutamate systems. In MD, dysregulated genes includesgenes involved in transmission of nerve impulses, neurogenesis, and thefibroblast growth factor system (FGF). (see FIGS. 10-12). Gene ontology(i.e., genetic signatures) for BP and MD can conveniently be used indeveloping diagnostic and therapeutic regiments for mood disorders.

Example 3 Identification of Additional Genes Dysregulated in MoodDisorders Using Rat Models of Depression and Anti-Depressant Treatment

Rats were exposed to chronic unpredictable stress treatments in parallelwith chronic anti-depressants treatment (e.g., the tricyclicantidepressant desipramine and the specific serotonin reuptake inhibitorfluoxetine). Saline treated stressed rats (SS) and saline treatednon-stressed rats (SN) were used as controls. In particular, salinetreated stressed rats (SS) were compared to desipramine treated stressedrats (DS); saline treated stressed rats (SS) were compared to fluoxetinetreated stressed rats (FS); saline treated non-stressed rats (SN) werecompared to desipramine treated non-stressed rats (DN); saline treatednon-stressed rats (SN) were compared to fluoxetine treated non-stressedrats (FN); and saline treated stressed rats (SS) were compared to salinetreated non-stressed rats (SN). Gene expression changes in rat cortexfollowing treatment were measured. The genes identified in this studyare shown in Table 6. This data suggests that different classes ofantidepressants, i.e., antidepressants with apparently differentmechanisms of action may act through a common biochemical pathway.

The above examples are provided to illustrate the invention but not tolimit its scope. Other variants of the invention will be readilyapparent to one of ordinary skill in the art and are encompassed by theappended claims. All publications, databases, Genbank sequences, GOterms, patents, and patent applications cited herein are herebyincorporated by reference.

TABLE 1 GenBank DLPFC-MDD Chromosome Accession# Gene Description OMIMLocation NM1964 Early growth response protein 1 (EGR1) EGR1 5q31.1 NM599human Insulin-like growth factor binding protein IGFBP5 2q33-34 5(IGFBP5) M87771 Fibroblast growth factor receptor k-sam, Splicek-sam-III 10q26 3 (k-sam-III) Z24725 H sapiens Mitogen-inducible gene(mig-2) mig-2 14q22.1 M64347 human Novel growth factor receptor (FGFR3)FGFR3 4p16.3 M80634 human Keratinocyte growth factor receptor FGFR210q26 (FGFR2) (SEQ ID NO: 1) Z14228 Nuclear mitotic apparatus protein 1,Alt. Splice NUMA U4 11q13 Form 2 (NuMA Clone U4) X67951 humanProliferation-associated gene (PAGA) PAGA 1p34.1 GenBank DLPFC-MDDAccession # Gene Description AF036268 SH3-domain GRB2-like 2 OMIM - SH3DOMAIN, GRB2-LIKE, 2; SH3GL2 AF060877 regulator of G-protein signalling20 OMIM - REGULATOR OF G PROTEIN SIGNALING 20; RGS20 AL049538 rasassociation (RalGDS/AF-6) domain containing OMIM - RAL GUANINENUCLEOTIDE DISSOCIATION STIMULATOR; protein JC265 RALGDS D14838fibroblast growth factor 9 (glia-activating factor) OMIM - FIBROBLASTGROWTH FACTOR 9; FGF9 D26070 inositol 1,4,5-triphosphate receptor, type1 OMIM - INOSITOL 1,4,5-TRIPHOSPHATE RECEPTOR, TYPE 1; ITPR1 J02902protein phosphatase 2 (formerly 2A), regulatory OMIM - PROTEINPHOSPHATASE 2, STRUCTURAL/REGULATORY subunit A (PR 65), alpha isoformSUBUNIT A, ALPHA; PPP2R1A J04513 fibroblast growth factor 2 (basic)OMIM - FIBROBLAST GROWTH FACTOR 2; FGF2 L05624 mitogen-activated proteinkinase kinase 1 OMIM - MITOGEN-ACTIVATED PROTEIN KINASE KINASE 1; MAP2K1M64788 RAP1, GTPase activating protein 1 OMIM - RAP1, GTPase-ACTIVATINGPROTEIN 1; RAP1GA1 M87771 fibroblast growth factor receptor 2 (bacteria-OMIM - FIBROBLAST GROWTH FACTOR RECEPTOR 2; FGFR2 expressed kinase,keratinocyte growth factor receptor, craniofacial dysostosis 1, Crouzonsyndrome, Pfeiffer syndrome, Jackson- Weiss syndrome) M96995 growthfactor receptor-bound protein 2 OMIM - GROWTH FACTOR RECEPTOR-BOUNDPROTEIN 2; GRB2 U09759 mitogen-activated protein kinase 9 OMIM -MITOGEN-ACTIVATED PROTEIN KINASE 9; MAPK9 U24152p21/Cdc42/Rac1-activated kinase 1 (STE OMIM - p21/CDC42/RAC1-ACTIVATEDKINASE 1; PAK1 20 homolog, yeast) U49857 transcriptional activator ofthe c-fos promoter W28432 Cluster Incl. W28432: 47f2 Homo sapiens OMIM -NEUROTROPHIC TYROSINE KINASE, RECEPTOR, TYPE 2; NTRK2 cDNA /gb = W28432/gi = 1308443/ug = Hs.92030 /len = 921 X07109 protein kinase C, beta 1OMIM - PROTEIN KINASE C, BETA-1; PRKCB1 X54938 inositol1,4,5-trisphosphate 3-kinase A OMIM - INOSITOL 1,4,5-TRISPHOSPHATE3-KINASE A; ITPKA Z71929 fibroblast growth factor receptor 2 (bacteria-OMIM - FIBROBLAST GROWTH FACTOR RECEPTOR 2; FGFR2 expressed kinase,keratinocyte growth factor receptor, craniofacial dysostosis 1, Crouzonsyndrome, Pfeiffer syndrome, Jackson- Weiss syndrome) GenBank Antcg BPAccession # Description Symbol NM_004794 RAB33A, member RAS oncogenefamily RAB33A NM_002844 protein tyrosine phosphatase, receptor type, KPTPRK M14752 M14752 HUMABLA Human c-abl gene |Gen ABL1 Bank==M14752NM_005252 v-fos FBJ murine osteosarcoma viral oncogene FOS homologNM_002229 jun B proto-oncogene JUNB NM_014813 KIAA0806 gene productKIAA0806 AB007943 AB007943: Homo sapiens mRNA for KIAA0474 RAP1GA1protein |GenBank==AB007943 NM_004067 chimerin (chimaerin) 2 CHN2NM_003676 degenerative spermatocyte homolog, lipid DEGS desaturase(Drosophila) NM_000830 glutamate receptor, ionotropic, kainate 1 GRIK1NM_002487 necdin homolog (mouse) NDN NM_002921 retinal G protein coupledreceptor RGR NM_001390 dystrobrevin, alpha DTNA NM_006000 tubulin, alpha1 (testis specific) TUBA1 NM_001634 S-adenosylmethionine decarboxylase 1AMD1 NM_006931 solute carrier family 2 (facilitated glucosetransporter), SLC2A3 member 3 NM_003832 phosphoserine phosphatase-likePSPHL NM_005010 neuronal cell adhesion molecule NRCAM NM_002073 guaninenucleotide binding protein (G protein), GNAZ alpha z polypeptide L24123L24123: Homo sapiens NRF1 protein (NRF1) NFE2L1 mRNA/cds = UNKNOWN /gb =L24123 /gi = 438646 /ug = Hs.83469 /len = 4992|Gen Bank==L24123NM_000810 gamma-aminobutyric acid (GABA) A receptor, GABRA5 alpha 5NM_005398 protein phosphatase 1, regulatory (inhibitor) PPP1R3C subunit3C AI526089 AI526089: DU3.2-7.H07.r Homo sapiens cDNA| COX5BGenBank==AI526089 NM_000840 glutamate receptor, metabotropic 3 GRM3NM_012249 ras-like protein TC10 TC10 NM_004791 integrin, beta-like 1(with EGF-like repeat ITGBL1 domains) NM_000615 neural cell adhesionmolecule 1 NCAM1 NM_003916 adaptor-related protein complex 1, sigmaAP1S2 2 subunit NM_001406 ephrin-B3 EFNB3 NM_001718 bone morphogeneticprotein 6 BMP6 X66358 X66358 cds#2 HSSTHPKB H. sapiens mRNA CDKL1KKIALRE for serine/threonine protein kinase|GenBank==X66358 DLPC-BPD00654 actin, gamma 2, smooth muscle, enteric ACTG2 U19599 U19599HSU19599 Human (BAX delta) mRNA| BAX GenBank==U19599 NM_006908ras-related C3 botulinum toxin substrate 1 RAC1 (rho family, small GTPbinding protein Rac1) NM_002374 microtubule-associated protein 2 MAP2AJ001612 phosphoserine phosphatase-like PSPHL NM_000293 phosphorylasekinase, beta PHKB NM_020217 hypothetical protein DKFZp547I014DKFZp547I014 NM_004379 cAMP responsive element binding protein 1 CREB1NM_032041 neurocalcin delta NCALD NM_015716 Misshapen/NIK-related kinaseMINK AF059274 Homo sapiens cDNA FLJ37320 fis, clone CSPG5 BRAMY2018106NM_006158 neurofilament, light polypeptide 68 kDa NEFL NM_002730 proteinkinase, cAMP-dependent, catalytic, PRKACA alpha NM_003885cyclin-dependent kinase 5, regulatory sub CDK5R1 unit 1 (p35) NM_003020Secretory granule, neuroendocrine protein 1 (SGNE1)(7B2 protein) locatedat chromosome band 15q13

TABLE 2 NM1964 Early growth response protein 1 (EGR1) NM599 humaninsulin-like growth factor binding protein 5 (IGFBP5) M87771 Fibroblastgrowth factor receptor k-sam, Splice 3 (k-sam-III) Z24725 H sapiensMitogen-inducible gene (mig-2) M64347 human Novel growth factor receptor(FGFR3) M80634 human Keratinocyte growth factor receptor (FGFR2) (SEQ IDNO: 1) Z14228 Nuclear mitotic apparatus protein 1, Alt. Splice Form 2(NuMA Clone U4) X67951 human Proliferation-associated gene (PAGA)AF036268 SH3-domain GRB2-like 2 AF060877 regulator of G-proteinsignalling 20 AL049538 ras association (RaIGDS/AF-6) domain containingprotein JC265 D14838 fibroblast growth factor 9 (glia-activating factor)D26070 inositol 1,4,5-triphosphate receptor, type 1 J02902 proteinphosphatase 2 (formerly 2A), regulatory subunit A (PR 65), alpha isoformJ04513 fibroblast growth factor 2 (basic) L05624 mitogen-activatedprotein kinase kinase 1 M64788 RAP1, GTPase activating protein 1 M87771fibroblast growth factor receptor 2 (bacteria-expressed kinase,keratinocyte growth factor receptor, craniofacial dysostosis 1, Crouzonsyndrome, Pfeiffer syndrome, Jackson-Weiss syndrome) M96995 growthfactor receptor-bound protein 2 U09759 mitogen-activated protein kinase9 U24152 p21/Cdc42/Rac1-activated kinase 1 (STE20 homolog, yeast) U49857transcriptional activator of the c-fos promoter W28432 Cluster Incl.W28432: 47f2 Homo sapiens cDNA /gb = W28432 /gi = 1308443 /ug = Hs.92030/len = 921 X07109 protein kinase C, beta 1 X54938 inositol1,4,5-trisphosphate 3-kinase A Z71929 fibroblast growth factor receptor2 (bacteria-expressed kinase, keratinocyte growth factor receptor,craniofacial dysostosis 1, Crouzon syndrome, Pfeiffer syndrome,Jackson-Weiss syndrome) NM_004794 RAB33A, member RAS oncogene familyNM_002844 protein tyrosine phosphatase, receptor type, K M14752 M14752HUMABLA Human c-abl gene|GenBank==M14752 NM_005252 v-fos FBJ murineosteosarcoma viral oncogene homolog NM_002229 jun B proto-oncogeneNM_014813 KIAA0806 gene product AB007943 AB007943: Homo sapiens mRNA forKIAA0474 protein|GenBank==AB007943 NM_004067 chimerin (chimaerin) 2NM_003676 degenerative spermatocyte homolog, lipid desaturase(Drosophila) NM_000830 glutamate receptor, ionotropic, kainate 1NM_002487 necdin homolog (mouse) NM_002921 retinal G protein coupledreceptor NM_001390 dystrobrevin, alpha NM_006000 tubulin, alpha 1(testis specific) NM_001634 S-adenosylmethionine decarboxylase 1NM_006931 solute carrier family 2 (facilitated glucose transporter),member 3 NM_003832 phosphoserine phosphatase-like NM_005010 neuronalcell adhesion molecule NM_002073 guanine nucleotide binding protein (Gprotein), alpha z polypeptide L24123 L24123: Homo sapiens NRF1 protein(NRF1) mRNA /cds = UNKNOWN /gb = L24123 /gi = 438646 /ug = Hs.83469 /len= 4992|GenBank==L24123 NM_000810 gamma-aminobutyric acid (GABA) Areceptor, alpha 5 NM_005398 protein phosphatase 1, regulatory(inhibitor) subunit 3C AI526089 AI526089: DU3.2-7.H07.r Homo sapienscDNA|GenBank==AI526089 NM_000840 glutamate receptor, metabotropic 3NM_012249 ras-like protein TC10 NM_004791 integrin, beta-like 1 (withEGF-like repeat domains) NM_000615 neural cell adhesion molecule 1NM_003916 adaptor-related protein complex 1, sigma 2 subunit NM_001406ephrin-B3 NM_001718 bone morphogenetic protein 6 X66358 X66358 cds#2HSSTHPKB H. sapiens mRNA KKIALRE for serine/threonine proteinkinase|GenBank==X66358 D00654 actin, gamma 2, smooth muscle, entericU19599 U19599 HSU19599 Human (BAX delta) mRNA|GenBank==U19599 NM_006908ras-related C3 botulinum toxin substrate 1 (rho family, small GTPbinding protein Rac1) NM_002374 microtubule-associated protein 2AJ001612 phosphoserine phosphatase-like NM_000293 phosphorylase kinase,beta NM_020217 hypothetical protein DKFZp547I014 NM_004379 cAMPresponsive element binding protein 1 NM_032041 neurocalcin deltaNM_015716 Misshapen/NIK-related kinase AF059274 Homo sapiens cDNAFLJ37320 fis, clone BRAMY2018106 NM_006158 neurofilament, lightpolypeptide 68 kDa NM_002730 protein kinase, cAMP-dependent, catalytic,alpha NM_003885 cyclin-dependent kinase 5, regulatory subunit 1 (p35)

TABLE 3 Acc. Disorder/Region Description Numb. MD DLPFC carboxypeptidaseD U65090

prostaglandin D2 synthase (21 kD, brain) AI207842

NEL-like 1 (chicken) D83017

zinc finger protein 36, C3H type-like 1 X79067

phosphoribosyl pyrophosphate synthetase 1 X15331

MD AnCng solute carrier family 1 (glial high affinity glutamatetransporter), member 3 D26443

clathrin, light polypeptide (Lcb) M20470

aldolase A, fructose-bisphosphate X05236

ubiquitin carboxyl-terminal esterase L1 (ubiquitin thiolesterase) X04741

BP AnCng v-raf-1 murine leukemia viral oncogene homolog 1 X03484

cytochrome c oxidase subunit Vb AI526089

proteasome (prosome, macropain) 26S subunit, non-ATPase, 1 D44466

tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation X56468

protein, theta polypeptide nuclear receptor subfamily 4, group A, member1 L13740

chondroitin sulfate proteoglycan 3 (neurocan) AF02654

fatty acid binding protein 7, brain AJ00296

BP DLPFC carboxypeptidase D U65090

indicates data missing or illegible when filed

TABLE 4 Summary Genbank Accession No. AnCg BP AnCg MD DLPFC BP DLPFC MDCB BP CB MD flags D50310 up up 1 L08485 up 1, 2, 3 U28964 up 1 AF016917up up 1, 3 L19182 down 1 AJ001612 up down up down up 1, 3 U66879 up 1J04046 up 1 X63575 up up up 1, 3 S74445 up 1, 2, 3 X71490 up up up up 1AF112471 up up 1, 2, 3 AB006626 up 1 U37143 down down 1, 3 AC004131 updown up down down 1 M29273 down up down 1, 2 X76220 up up 1, 2 M12267 up3 AF060877 down down down 1 AB018305 down 1, 2 U58334 down down down 1AB020629 up down up down 1, 3 U37122 down down 1, 3 AL080061 down downdown down 1, 3 M34309 down down 1 M80634 down down 1 (SEO ID NO: 1)M64347 down down 1 X57206 down down down 1, 3 X77196 down down 1, 3Z24725 down down down 1 AB018342 down down 1, 3 Y10275 down up 1, 3AB007943 down 1 AL049538 down down down 1 M14758 down down down 3 X13839down down 3 X63432 up up 3 X04098 up up up 3 AF006082 up down 3 D67031down down down 3 L22214 up 3 J03473 up 3 AJ236876 up up up AF072902 upup U84011 down down K02215 up up up AI800578 down down down AL049954down R59606 down down M80899 down down 2, 3 U00957 up up AA114830 upU81607 up 3 M90360 down down X15414 up up 3 U05861 up D17793 down down 3K03000 down down 2 U46689 up down 3 U24267 down down down M93405 downdown 3 U34252 up up 3 X05236 up up up up up M21154 up up up 3 W63793 up3 M63175 up up AB028994 down down U29926 down X81438 up up up up 3D14662 down down down 3 AF091077 down 3 X97074 up up up up 2 D38293 upup up 3 J02611 down down M12529 down down 3 D86981 up down 3 U41518 downdown U34846 down down up 2, 3 D87468 down 2 L04510 up 2, 3 AF049884 up 3U02570 down down AB002292 down U50523 up 3 AI525393 up 3 AF006087 up up2, 3 AF006088 up 3 Z11501 up L08424 down down 3 M27396 up up 3 S67156down down AL096842 down down AB018258 down J05096 down down down 2M37457 up up 2 W28508 down up AF007876 down down J04027 up 2, 3 L20977up up 3 W28589 up up 3 AJ010953 up 2 D14710 up up up up 3 U09813 up up 3AF087135 up up 3 AA845575 up up 3 AF047436 up up 3 AA917672 up 3 X83218up up 3 D16469 up up AL049929 up up 3 D89052 up up 3 AI318615 down 3AI547262 up 3 AA056747 up 3 L09235 up up up 3 AA877795 up up 2 X76228 upup up up 3 W26326 up up up X79888 up 2 X66030 down M76125 down S82297down down AB021288 down down V00567 down down down AF029893 up up up up3 AF082868 down down 3 U50708 down U00115 down down AL049257 up downdown AB004066 down down down AF001383 up up 2, 3 U68485 up 2, 3 AF002697up up 3 S78771 up up up up 3 AC005306 down up AB023169 up up U72649 downdown down 2 AF047472 up down AB023171 up down down X94910 up up up up 3AF054175 up 3 AF014837 down down AF009425 down down down X95592 up 2, 3AB007948 up up up D86062 up 3 AL080097 down down down AF006621 up up up3 J03037 down up down down 3 U79666 down 3 M76559 up 2 S60415 up 3AF068862 up up up up 2 U12022 up up up D45887 up up AB020640 up U02390up up 2 U02390 up up U02390 up X85030 down down U20325 up 2 AB002376 updown AL035079 up up AF070648 down down L10822 up up AF091433 downAF026166 up up 3 X74801 up up 3 AF026292 up 3 X69398 up up M38690 updown down AF023158 down M37712 down AL031282 up down M35543 up up downW27541 down down U59325 up up AF006484 down down 3 X66364 up 2 L04658 upup up X77743 up up 3 X66358 up U22398 down down down M16965 up up upW27184 up up up U65887 down 3 U60808 down 3 AI056696 up 3 U78516 downAL080084 up up 3 AA189161 up up AB023203 down down 3 U03749 up 2 Y00064up U07223 up up 2 X70297 up W29042 up 3 D49738 up 3 Z30644 down 3 U89916down down M59287 down down down M59287 down down down AF039704 up X91788up 3 M20469 up up up 3 M20470 up up up up 3 AB020709 up up 2, 3 S80562down 2, 3 D13146 up up M19650 up 3 Z21488 up up AB020675 up AB014533 updown down M92642 down down M58526 down down U65928 up up up 3 AA149486up up 3 M22760 up 3 M19961 up 3 AI526089 up up up 3 T57872 up up 3AA152406 down 3 AA978033 up 3 AB007618 up 3 N50520 up up 3 U65090 updown X51405 up S74445 up 2, 3 M27691 down 2 S68271 up up 3 D10656 up up3 U49857 down down U49857 down down down AL038340 up up AL038340 up upAF039397 up up AF053641 up 3 M27826 down U89896 down up up 3 D32039 downup X15998 down X15998 down AF026547 down down down M33146 down 2 L22569up up X16832 down down 3 Y07593 up up up L06797 down down 3 L47738 up upup M33318 down 3 Y11307 down down down M98529 up up up up up AB002379down down down D15057 up 3 AL050152 up down D31767 up 3 AL050084 downdown AB002367 up down down 3 AF086947 up up U50733 up up up 3 W26651 upup up U48705 down L20817 down down U59321 down up AF000982 up AF007142down down U63825 up up 3 AF021819 up 3 AL080115 up down AL049944 up upAL049934 down down down down AL050390 up up AL050272 up up AL050159 downdown L08069 up up up up L08069 up up up AI810807 up up 2, 3 AI540958 up3 AF000430 up up up 3 D50857 down down down 3 AF007875 up 3 U97105 upM97388 up up 3 D83407 up up S65738 up 3 U26742 down 3 U46744 down up 3U84551 down up U46746 down 3 X68277 down down down 3 L05147 up AB013382down down 3 U31930 up 3 U46461 down 3 D86550 up 2, 3 M91670 up up M91670up up AL050282 up up M31210 down D13168 down X70940 up up 3 AB023159 upup up up up up U03877 down down U66406 up up 3 AB011542 up L18960 downAF035280 up 3 U36764 up 3 U39067 up up up 3 U94855 up up 3 U54558 up 3AC002544 down down D13748 up up L36055 down 3 U49436 up up 2,3 AL080199down down C18655 down AB002303 down down X51956 up 2 L35594 down up downdown 2, 3 L35594 down up down down 2, 3 D45421 down 2 AF103905 downU81984 down 3 D83492 up up U12535 down M34309 down down down 3 X81625 upup down 3 J04058 up 3 AB028990 up J02931 down AJ002962 up up 3 AA977580up 2 W26480 down down down AF035284 down down X87241 down down AF000561down M30448 up up up AB014596 up 3 D14697 up up 3 U60060 up 3 U60061 up3 X59065 down down down Z70276 up up up 3 U66198 up 3 D14838 up 2 Z69641down down down Z69641 down M87770 down down down Z71929 down down downX55741 up up up AF070557 up W27472 down down W26655 up up AF052106 up upAL049949 down down down down 3 X02761 down down V01512 down down downV01512 down down down down K00650 down down AF032885 down down M84562down U32519 up 3 AB014560 up 3 AI565760 up 3 AJ225028 up 3 M82919 up up3 X15376 up up 3 D86181 up down S68805 down D00723 up down 3 Y13286 upup 3 S40719 down down down 3 D87467 up M65188 down 3 X52947 down downdown 3 M57609 down down 3 U33267 up up up 3 X76648 up 3 AB020645 up up2, 3 U08997 up down U08997 down X59834 down down down 3 U43083 up up 3D90150 up up AF017656 up up up AJ238764 up 3 AL049367 down AB020662 downdown down down AF047438 down 3 M22632 up up 3 AF016004 down up down down3 D38449 up 3 U87460 up down up down down AJ011001 down X71973 up 2, 3W28944 up up 3 M81886 up up 3 U10301 up 3 X82068 up 3 L19058 down 3S40369 up up X77748 up up 2, 3 D87119 down down down down 3 D87119 downdown down down 3 X04412 up down up down down 3 M16594 down down J05459up 2, 3 U90313 up up up 3 M95809 up 3 X03473 up D64142 up D64142 upL19779 up M37583 up up 3 H15872 up up AA255502 down up up M25079 downdown down L48215 down down AF019214 down down down AF029890 up up upU31814 up 3 AL034374 down up down down AI391567 down 3 U51004 up 3AB014555 down 3 X58536 down down M32578 down U23803 up 3 X12671 upM16342 up 3 D89678 up 3 U01923 up 3 W27191 down 3 X92814 up 2 X99209 upAF068754 up 3 M11717 down down down L26336 up up up up up L26336 up upup up L26336 up down up down down L26336 up down L12723 up 2 X87949 up 3X13794 up up up Y00371 up up up up L15189 up up 2, 3 AL021937 up X15183up 3 J04988 up up up W28616 up M22382 up 3 AI912041 up 2, 3 X57830 up 2,3 AI434146 down 3 AF012023 up X77956 down down AL022726 down U49283 upup up AA522698 up up up 3 X17025 up 3 U66042 up up M24594 up 3 X16302 upup AB017563 down 2 L42572 up 3 U26398 up up X77567 up up up 3 U96876 up2 X53586 down down X07979 down 2 AL021786 down AA477898 up up X54938 upup up up 3 U23850 up up 3 AB016492 up up J04111 up up X51345 down downdown AF070523 up D79994 down down down L02840 down 3 U52155 down 3U39196 up Y15065 up 3 D26067 up 3 D31887 up up 3 D14663 up 3 AL049250down down down D87074 up down D87443 up down 3 D87445 down AB002347 upAB002361 up AB007903 up 3 AB007963 down down AB011095 down down AB014526down down AB014544 down down down down AB018335 down down AB020637 upAB020661 down down AB023152 up AB023209 down down AB023230 down down 3AB028972 up up up AB028977 down 3 AB029034 down down AF070621 up 3Y08319 up 3 AB002357 up 3 AF035621 up up up 3 U59919 up 3 AJ001685 downdown J04182 up up down 3 U36336 down up down down 3 Y11395 up upAL050126 down 2 M90424 down 3 X02152 up up 3 X13794 up up AI535946 up upup up D55696 up 3 AF087693 up 2 X76488 up up down 3 U41060 up 3 X61118down U79297 up up AL039458 down down 2 AB011540 down M63959 up up up up3 M92439 up up up up AB012293 up up up 3 W26633 up up U03100 up up down3 U03100 up down 3 D55649 up up 3 AA420624 down 3 U01828 up up U89330 upup S76756 down 3 L05624 up up up U17743 up up up 3 U71087 up up 3 Z11695up up up 3 X14474 up 2, 3 X66867 down AF072250 up down D84557 up downX79440 up 3 S57212 up AW006742 down down down AI674208 down down upAI674208 down down up AF038186 up W26659 up AB014579 up up M16279 down 2D25217 down AI127424 up 3 AF001359 down X70326 down up down 3 AF041080down down down down AI670788 up up Z48051 up down U64565 down up 3D14812 up 2, 3 AI597616 up up 3 Y11681 up up up up up 3 AL050361 up 3Z98946 down AI547258 down down 2 AF072928 up up down 3 M55405 down downAF001548 down down down 2 AF013570 down down 2, 3 AF001548 down downdown 2 D10667 down 2, 3 D10667 down 2, 3 J02854 down down 3 AF020267down 3 AB029029 up down U42349 up up up 3 AF052142 up up up 3 AA126505up 3 X77548 down 2 AF044209 up up U35139 up up up 2, 3 D87953 down downAF047185 up 3 AC002400 up up up AI345944 up 3 AA203354 up 2, 3 AF047181up up up 3 AA527880 up 3 AA760866 up 3 AF050640 up up 3 AI541336 up 3AC005329 up up AF053070 up up up up 3 Y16241 down down 3 D63878 downdown down D23662 up X05608 up up up D83017 up up up 3 W27762 down down 3X64318 up 3 Z83840 up up up AB023192 up up up up 3 AF019415 down downAF019415 down down X17620 up 2, 3 X73066 up up up 2, 3 AL038662 up 2, 3X58965 up 2, 3 M86707 up up up 2 AI816034 up up U97669 down down W28770up up up 3 AF002020 up down 3 AF002020 up down down down 3 AJ132583 upup 3 U61849 up up up up up up 2, 3 AI198311 up 2, 3 L13740 down downdown down down down L13740 down down down down down down AB002341 updown 2 U55258 up up 2 U55258 up up 2 X99076 up AB011150 up up up up 3U03985 up up 2, 3 X55740 down AI018523 down down down X75958 down downup down 2, 3 W28432 down down down Y10148 down down AL050066 up up upU48250 down down down U48250 down M63623 up down AF061034 up up 3AF061034 up 3 U63717 up up 3 U62961 up 3 AB017016 up up up M80482 updown AB023211 down down down down L13385 up 2, 3 D63391 up up 3 U24152up up up up 2, 3 U24152 up up up 2, 3 AF068864 up up up up AF068864 upup up up AF005043 up up 3 M93650 down 3 AI521453 up up up 3 X73424 up up3 AB020631 down down D13892 up up 3 D25547 up up up up up 3 U52969 up up3 AA535884 down U40370 up 2 AB007946 up S41458 down AF056490 down downdown 3 L42451 up 3 X98248 up up up AB002345 down down down 2 AF093670 updown 3 U41816 down 3 AL096719 up 3 V00572 up up up 2, 3 M83088 up 2, 3X84908 up U45976 up down 3 AF010312 up down up down AL120815 up up downdown down Z29090 down 3 U81802 up up 3 U49070 up up W28299 up up upAL050371 up D30037 up U03090 down U60644 up up up 3 U84573 down 3 M54927down up down down 3 M22299 up down 3 X57398 up up up up up D11428 updown up down down 3 AF001601 down down AL050161 down down down AF017786down down up 2 AF016371 up 3 AF001691 up down 3 Y18207 down down Y18207down N36638 down down down 3 Z50749 up 3 J02902 up J02902 up M64929 upup M64929 up M29551 up 2 X89416 up up U44772 up 3 AB014512 down 3 X67951up 2 L19185 up up up U25182 up up M33336 up up 3 M33336 up up 3 M33336up X07109 up up 3 X06318 up up 3 Z15108 up up 3 U29185 up 2 AB011124 upup up X15331 up up up 3 D87258 down down down D87258 down down downJ03077 up up 3 M85169 up up 3 L76517 up up down 3 D00760 up 2, 3 D00762up up 3 D00761 up up 3 D26598 up 2, 3 D26600 up up 3 D26600 up up 3D29011 up up 2 D29012 up 3 D38048 up up up 3 AF035309 up D44466 up up 2,3 AL031177 up up AB009398 up 2, 3 D78151 up 3 U51007 up D50063 up 3D38047 up up up up 3 D38047 up 3 AJ001612 down up down up down up 3D14694 up M98539 down down down AI207842 down down 3 AI207842 down down3 U33284 up M14630 down M57399 down down 2, 3 M57399 down down down 2, 3X54131 down L77886 up Z48541 up up 2 D64053 up M93426 down down down 2,3 X63578 up 2 Z48054 down 3 AL031781 down AL031781 down down downAI540957 up 3 AF052113 up up 3 M28209 up up 3 AL050268 up 3 AL050268 up3 U59877 down down down 3 AI189226 down down D14889 up up 3 D14889 up upup 3 M28212 up up 3 AJ133534 up up 3 X98001 up D25274 up U41654 up 2, 3M35416 up M31469 up up up up up M31469 up up up up AF054183 up up up upup 3 X63465 up up X63465 up S80343 up 3 D79990 down down down U28686down down down down down U89505 up up U23946 up M11433 down 3 X00129 upup up 3 N92548 down down AW044624 up 3 U27768 up 2, 3 U78166 up up 3D26129 up up down down AF037204 up down 3 X13973 up up M63488 up up upD87735 up 2, 3 X55954 up up 3 AI708983 up up 3 X57958 up 3 Y00281 up up2, 3 AL031659 up up AA977163 up up up 3 M13932 up 3 AI526078 up 3 D14530up 3 S79522 up 3 X55715 up up 3 M84711 up up up 2, 3 Y11651 up 3 L10333up up up 2, 3 AB020693 up up M84820 up AL049940 up up down AJ001515 downAB020658 up X91257 up up up up 3 M55580 down down AF051323 up up upD12676 up up 3 M25756 up down down 2 AF070614 up 3 L10338 down 3AF049498 up up up up 2, 3 AB011178 down down down AB007937 down downAB015345 up up up up X97064 up 3 AJ131245 up 3 AF055006 up 3 AF054184 upup 2, 3 U73167 down down AB000220 up AB002438 down down down Z11793 downD86957 down down down 3 AI743134 down down down Z81326 up 3 D28423 up upAL031681 up down L41887 up down Y00757 up up 2 AF036268 up up up 3AB007960 down down U33760 up up up up W26700 up up up U08989 up 3 U01824down down 3 W28850 down 3 D26443 down down down 3 H10201 down X60036 up3 M20681 up up 3 AF007216 down down down 2, 3 AF011390 down up 2, 3AF015926 down down 3 D86959 up down U96094 up up up 3 D80000 up down 2,3 X59960 up up 3 AF053136 up 2 AL049650 up up up AA733050 up up U40571down 3 AF034546 up 3 X02317 up 3 X63753 up down 3 AJ001183 down 2 Z46629down down down AB011088 down down J03040 down down J04765 up up AF052124up up down AF039843 down down down Y08685 up down 3 D78130 up 3 M32313up 2 M32886 down down 3 U88666 up 3 J00306 up 2 AI636761 up 2, 3AB011107 down down down L78440 up up U04735 up up up up M86752 up upX99325 up up up up 2, 3 AF099989 up down 3 M31303 up 2 X85116 down downdown down AL035306 up 3 U77942 down 2 D63506 down down U34804 up U40215up up up up 3 AF039945 down 2 U93305 up up X68194 down up down down 3D38522 up U18062 up up 3 M95787 down down AF010400 up up AL050265 up 3AL050107 down down D50495 up 2 M80627 down down down D15050 down downdown U19969 down down X52882 up 3 U49188 up up L24804 up X75861 up 3W28869 up up 3 L06139 down down down X93512 up up S95936 up up down down3 M55153 down 3 L12350 down L12350 down down AJ133115 down down M24748up up X97544 up 3 X97544 up 3 L27476 down down AB028950 down AI688299down up down down 3 R16035 up 3 U81006 up 3 M92383 up D38305 down downD13641 up 3 U09477 up 3 M12125 down 3 M12125 down down 3 U12595 up 3X00437 down down AB011089 up 3 AF084260 up 3 AJ133769 up up X89066 up 3AF042181 up down up AF001294 up AF035283 down down X06956 up up up upX06956 up up up 2 AF005392 up up up X01703 up up AF035316 up down down 3X02344 up down U47634 up 3 X00734 up up S75463 up 3 D17517 up downU18934 up AI310002 up 2 AF075599 up up up up U67122 up 3 X04741 up up up3 U27460 up up U30930 down up down 3 T79616 up 3 J04973 up up 2, 3L32977 up AA526497 up 3 U30888 up M36200 up up AL050223 up 3 U56833 up 3L06132 up 3 AJ002428 up up up up L08666 up 3 AF024710 down down AF022375down down M63978 down up X51521 down Z19554 down down down 3 AF060902 upup D26068 down 2 AB011113 up down W27944 down down W26496 down down downD14661 up Y08614 up 3 J04977 up up up 3 M30938 up up U89436 up up 3X56468 up 3 X56468 up up 3 M92843 down down down 2, 3 U07802 down downX78992 down down down AL050276 down down L11672 down AD000092 up upV00599 up down X55989 down S81916 up up up up J00153 down down downAL049423 down down AF052148 down down AL022101 up up up AL118582 downdown AI095508 up 2 W28612 down down down AL049378 down down AI700633down up down AF070536 down down AF052119 up up up AL080113 up AL049265down AL049390 down down AF070577 up up 2 AI827895 down down X95677 up upAL080093 up AL049969 down down down AF052141 up up up L27560 down W27522up AL022718 up down AJ005694 down 3 AL120687 up up AL046322 up upAW043812 down H12054 up AC003007 up up J03071 down down M57417 down downdown M33764 up M58028 up X74262 up U19796 up U22028 down X79568 downM55914 up M21154 up M10905 down U33429 up AB014539 down X63432 up upX56841 down Y00067 up AF007140 up X13839 down down AF023268 up AF053356down U37122 down down AB000450 up AI126004 up AF002668 up X54304 downU57843 down X02344 up X04098 up up up U96074 up D32053 up up U59632 downX14346 down Z98046 up AL096737 down AB014598 down U17886 up AI986201 upAL080181 down AB014514 up R92331 down U24183 up D00860 up U09510 upAI635895 up U66033 down U51334 down AF020762 up U24105 up M36820 downU59912 up X63368 up AF047863 up U11861 up AL080122 up M14648 down Y14153up X81637 down M88108 up AF042384 up AA704137 up AB011156 up AI862521 upAF047469 up AF025887 up AF091085 up AL035494 up AI540925 down D32129down AB028972 up AF091071 up AL040137 down X15187 up U48730 down L08488up K03460 up AF005361 up M95585 up M91670 up

TABLE 5 RT-PCR Confirmation Gene Bank AnCg DLPFC DLPFC Acc. No. GeneName BP AnCg MD BP MD AB020629 ABCA8 down U37122 ADD3 down X63575 ATP2B2down X71490 ATP6V0D1 up U66879 BAD down J04046 CALM3 up AF112471 CAMK2Bup D50310 CCNI up AL080061 CLIC4 down S74445 CRABP1 up up U37143 CYP2J2down down M34309 ERBB3 down M80634 FGFR2 down (SEQ ID NO: 1) M64347FGFR3 down L08485 GABRA5 up AF016917 GABRD down down AC004131 GPRC5Bdown down AB006626 HDAC4 up L19182 IGFBP7 down down X57206 ITPKB downX77196 LAMP2 down M29273 MAG down down X76220 MAL down down Z24725 MIG2down AB018342 MYO10 down M12267 OAT down down Y10275 PSPH down AJ001612PSPHL down down AB007943 RAP1GA1 down AF060877 RGS20 down down AL049538RIN2 down AB018305 SPON1 down down U58334 TP53BP2 down down U28964 YWHAZup

TABLE 6 Summary of Anti-Depressant Treatment Data Genbank Accession No.Gene Name SSvDS SSvFS SNvDN SNvFN SNvSS M80899 AHNAK down down K03000ALDH1A1 down down X97074 AP2S1 down U34846 AQP4 down down up D87468 ARCup down down down L04510 ARFD1 down AF006087 ARPC4 down J05096 ATP1A2down M37457 ATP1A3 down down up J04027 ATP2B1 up AJ010953 ATP2C1 downAA877795 ATP6V1D down X79888 AUH down AF001383 BIN1 down U68485 BIN1down U72649 BTG2 up X95592 C1D down M76559 CACNA2D1 up AF068862 CALB1down down AF112471 CAMK2B down U02390 CAP2 down down up U20325 CART upX66364 CDK5 down up U03749 CHGA down U07223 CHN2 down AB020709 CNK2 downS80562 CNN3 down S74445 CRABP1 up S74445 CRABP1 up M27691 CREB1 upM33146 CSRP1 down AI810807 DNCI1 down D86550 DYRK1A down U49436 EIF5down X51956 ENO2 up up L35594 ENPP2 down L35594 ENPP2 down D45421 ENPP2down AA977580 FACL3 down D14838 FGF9 up up L08485 GABRA5 down AB020645GLS down up down X71973 GPX4 up X77748 GRM3 down down J05459 GSTM3 downX92814 HRASLS3 down L12723 HSPA4 down down up L15189 HSPA9B up AI912041HSPE1 down X57830 HTR2A down down AB017563 IGSF4 up U96876 INSIG1 downX07979 ITGB1 down AL050126 LAP1B down down AF087693 LIN7A down downAL039458 LRIG1 up M29273 MAG up X76220 MAL down down X14474 MAPT upM16279 MIC2 down down D14812 MRGX down AI547258 MT2A down down downAF013570 MYH11 down down down down D10667 MYH11 down down down downD10667 MYH11 down down down down AF001548 MYH11 down down down downAF001548 MYH11 down down down down X77548 NCOA4 up up U35139 NDN downAA203354 NDUFB3 down X73066 NME1 down X17620 NME1 down AL038662 NME1down X58965 NME2 down M86707 NMT1 down U61849 NPTX1 down down AI198311NPY down U55258 NRCAM down AB002341 NRCAM down U55258 NRCAM down U03985NSF down X75958 NTRK2 down L13385 PAFAH1B1 down U24152 PAK1 down U24152PAK1 down U40370 PDE1A up AB002345 PER2 down down down V00572 PGK1 downM83088 PGM1 up AF017786 PPAP2B down M29551 PPP3CB down down X67951 PRDX1down U29185 PRNP up D00760 PSMA2 down D26598 PSMB3 down D29011 PSMB5 upD44466 PSMD1 up AB009398 PSMD13 down M57399 PTN down M57399 PTN downZ48541 PTPRO up M93426 PTPRZ1 down X63578 PVALB down up U41654 RAGA downU27768 RGS4 down down D87735 RPL14 down down Y00281 RPN1 down up M84711RPS3A down down L10333 RTN1 down up M25756 SCG2 down up AF049498 SCN2Bdown AF054184 SEC61G down Y00757 SGNE1 down AF007216 SLC4A4 down downAF011390 SLC4A4 down down D80000 SMC1L1 down AF053136 SNCB up AJ001183SOX10 up AB018305 SPON1 down M32313 SRD5A1 up AI636761 SST down up downJ00306 SST down up down X99325 STK25 down M31303 STMN1 down U77942 STX7down down up AF039945 SYNJ2 down D50495 TCEA2 up up X06956 TUBA1 downAI310002 UBE2D2 down J04973 UQCRC2 down D26068 WBSCR1 down M92843 ZFP36up AI095508 down down AF070577 down

TABLE 7a AnCg BP Genetic Ontology Genbank Accession No. Gene NameDescription 26S proteasome D00762 PSMA3 proteasome (prosome, macropain)subunit, alpha type, 3 D44466 PSMD1 proteasome (prosome, macropain) 26Ssubunit, non-ATPase, 1 D00761 PSMB1 proteasome (prosome, macropain)subunit, beta type, 1 D38048 PSMB7 proteasome (prosome, macropain)subunit, beta type, 7 D78151 PSMD2 proteasome (prosome, macropain) 26Ssubunit, non-ATPase, 2 D29012 PSMB6 proteasome (prosome, macropain)subunit, beta type, 6 D38047 PSMD8 proteasome (prosome, macropain) 26Ssubunit, non-ATPase, 8 D26598 PSMB3 proteasome (prosome, macropain)subunit, beta type, 3 D26600 PSMB4 proteasome (prosome, macropain)subunit, beta type, 4 synaptic transmission X82068 GRIA3 glutamatereceptor, ionotrophic, AMPA 3 D11428 PMP22 peripheral myelin protein 22AI636761 SST somatostatin AA126505 NCAM1 neural cell adhesion molecule 1L10338 SCN1B sodium channel, voltage-gated, type I, beta polypeptideX81438 AMPH amphiphysin (Stiff-Man syndrome with breast cancer 128 kDaautoantigen) M19650 CNP 2′,3′-cyclic nucleotide 3′ phosphodiesteraseL19058 GRIK1 glutamate receptor, ionotropic, kainate 1 AI198311 NPYneuropeptide Y U68485 BIN1 bridging integrator 1 M81886 GRIA1 glutamatereceptor, ionotropic, AMPA 1 Z11695 MAPK1 mitogen-activated proteinkinase 1 X77748 GRM3 glutamate receptor, metabotropic 3 AF052113 RAB14RAB14, member RAS oncogene family U40215 SYN2 synapsin II U61849 NPTX1neuronal pentraxin I Chaperone J04988 HSPCB heat shock 90 kDa protein 1,beta U12595 TRAP1 heat shock protein 75 L12723 HSPA4 heat shock 70 kDaprotein 4 L08069 DNAJA1 DnaJ (Hsp40) homolog, subfamily A, member 1AL038340 CRYAB crystallin, alpha B AL038340 CRYAB crystallin, alpha BX02344 TUBB2 tubulin, beta, 2 AF026166 CCT2 chaperonin containing TCP1,subunit 2 (beta) M63959 LRPAP1 low density lipoprotein receptor-relatedprotein associated protein 1 L26336 HSPA2 heat shock 70 kDa protein 2AF026292 CCT7 chaperonin containing TCP1, subunit 7 (eta) L08069 DNAJA1DnaJ (Hsp40) homolog, subfamily A, member 1 AA149486 COX17 COX17homolog, cytochrome c oxidase assembly protein (yeast) Y00371 HSPA8 heatshock 70 kDa protein 8 X74801 CCT3 chaperonin containing TCP1, subunit 3(gamma) X56468 YWHAQ tyrosine 3-monooxygenase/tryptophan 5-monooxygenaseactivation protein, theta polypeptide U41816 PFDN4 prefoldin 4 L15189HSPA9B heat shock 70 kDa protein 9B (mortalin-2) L26336 HSPA2 heat shock70 kDa protein 2

TABLE 7b AnCg MD: Genetic Ontology Genbank Accession Gene DescriptionNo. Name transporter activity T79616 UQCRB ubiquinol-cytochrome creductase binding protein AI526089 COX5B cytochrome c oxidase subunit VbAL049929 ATP6IP2 ATPase, H+ transporting, lysosomal interacting protein2 AF006621 C4orf1 chromosome 4 open reading frame 1 L09235 ATP6V1A1ATPase, H+ transporting, lysosomal 70 kDa, VI subunit A, isoform 1M22760 COX5A cytochrome c oxidase subunit Va U01824 SLC1A2 solutecarrier family 1 (glial high affinity glutamate transporter), member 2N50520 COX7B cytochrome c oxidase subunit VIIb AF007216 SLC4A4 solutecarrier family 4, sodium bicarbonate cotransporter, member 4 AF011390SLC4A4 solute carrier family 4, sodium bicarbonate cotransporter, member4 AA526497 UQCRH ubiquinol-cytochrome c reductase hinge protein AA845575ATP5J ATP synthase, H+ transporting, mitochondrial F0 complex, subunitF6 X52947 GJA1 gap junction protein, alpha 1, 43 kDa (connexin 43)D26443 SLC1A3 solute carrier family 1 (glial high affinity glutamatetransporter), member 3 AF053070 NDUFV1 NADH dehydrogenase (ubiquinone)flavoprotein 1, 51 kDa X76228 ATP6V1E1 ATPase, H+ transporting,lysosomal 31 kDa, VI subunit E isoform I X63575 ATP2B2 ATPase, Ca++transporting, plasma membrane 2

TABLE 7c DLPFC BP Genetic Ontology Genbank Accession No. Gene NameDescription hydrogen ion transporter activity AA917672 ATP5L ATPsynthase, H+ transporting, mitochondrial F0 complex, subunit g AI526089COX5B cytochrome c oxidase subunit Vb J04973 UQCRC2 ubiquinol-cytochromec reductase core protein II AF050640 NDUFS2 NADH dehydrogenase(ubiquinone) Fe—S protein 2, 49 kDa (NADH-coenzyme Q reductase) AL049929ATP6IP2 ATPase, H+ transporting, lysosomal interacting protein 2AF047181 NDUFB5 NADH dehydrogenase (ubiquinone) 1 beta subcomplex, 5, 16kDa AF047436 ATP5J2 ATP synthase, H+ transporting, mitochondrial F0complex, subunit f, isoform 2 D14710 ATP5A1 ATP synthase, H+transporting, mitochondrial F1 complex, alpha subunit, isoform 1 U09813ATP5G3 ATP synthase, H+ transporting, mitochondrial F0 complex, subunitc (subunit 9) isoform 3 N50520 COX7B cytochrome c oxidase subunit VIIbAF087135 ATP5H ATP synthase, H+ transporting, mitochondrial F0 complex,subunit d D89052 ATP6V0B ATPase, H+ transporting, lysosomal 21 kDa, V0subunit c″ X76228 ATP6V1E1 ATPase, H+ transporting, lysosomal 31 kDa, V1subunit E isoform 1 Chaperone U56833 VBP1 von Hippel-Lindau bindingprotein 1 L08069 DNAJA1 DnaJ (Hsp40) homolog, subfamily A, member 1AL038340 CRYAB crystallin, alpha B AL038340 CRYAB crystallin, alpha BX15183 HSPCA heat shock 90 kDa protein 1, alpha X56468 YWHAQ tyrosine3-monooxygenase/tryptophan 5-monooxygenase activation protein, thetapolypeptide L24804 TEBP unactive progesterone receptor, 23 kD W28616HSPCB heat shock 90 kDa protein 1, beta D49738 CKAP1cytoskeleton-associated protein 1 AF026166 CCT2 chaperonin containingTCP1, subunit 2 (beta) M63959 LRPAP1 low density lipoproteinreceptor-related protein associated protein 1 X87949 HSPA5 heat shock 70kDa protein 5 (glucose-regulated protein, 78 kDa) L26336 HSPA2 heatshock 70 kDa protein 2 M22382 HSPD1 heat shock 60 kDa protein 1(chaperonin) L08069 DNAJA1 DnaJ (Hsp40) homolog, subfamily A, member 1AF035316 TUBB tubulin, beta polypeptide AI912041 HSPE1 heat shock 10 kDaprotein 1 (chaperonin 10) Y00371 HSPA8 heat shock 70 kDa protein 8X74801 CCT3 chaperonin containing TCP1, subunit 3 (gamma) X56468 YWHAQtyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein,theta polypeptide W29042 CIA30 CGI-65 protein L15189 HSPA9B heat shock70 kDa protein 9B (mortalin-2) OXPHOS X71490 ATP6V0D1 ATPase, H+transporting, lysosomal 38 kDa, V0 subunit d isoform 1 D14710 ATP5A1 ATPsynthase, H+ transporting, mitochondrial F1 complex, alpha subunit,isoform 1, cardiac muscle U09813 ATP5G3 ATP synthase, H+ transporting,mitochondrial F0 complex, subunit c (subunit 9) isoform 3 AF087135 ATP5HATP synthase, H+ transporting, mitochondrial F0 complex, subunit dAA845575 ATP5J ATP synthase, H+ transporting, mitochondrial F0 complex,subunit F6 AF047436 ATP5J2 ATP synthase, H+ transporting, mitochondrialF0 complex, subunit f, isoform 2 AA917672 ATP5L ATP synthase, H+transporting, mitochondrial F0 complex, subunit g X83218 ATP5O ATPsynthase, H+ transporting, mitochondrial F1 complex, O subunit D89052ATP6V0B ATPase, H+ transporting, lysosomal 21 kDa, V0 subunit c″AA056747 ATP6V1A1 ATPase, H+ transporting, lysosomal 70 kDa, V1 subunitA X76228 ATP6V1E1 ATPase, H+ transporting, lysosomal 31 kDa, V1 subunitE isoform 1 AI526089 COX5B cytochrome c oxidase subunit Vb N50520 COX7Bcytochrome c oxidase subunit VIIb AC002400 NDUFAB1 NADH dehydrogenase(ubiquinone) 1, alpha/beta subcomplex, 1, 8 kDa AA203354 NDUFB3 NADHdehydrogenase (ubiquinone) 1 beta subcomplex, 3, 12 kDa AF047181 NDUFB5NADH dehydrogenase (ubiquinone) 1 beta subcomplex, 5, 16 kDa AF050640NDUFS2 NADH dehydrogenase (ubiquinone) Fe—S protein 2, 49 kDa(NADH-coenzyme Q reductase) J04973 UQCRC2 ubiquinol-cytochrome creductase core protein II L32977 UQCRFS1 ubiquinol-cytochrome creductase, Rieske iron-sulfur polypeptide 1 U17886 SDHB succinatedehydrogenase complex, subunit B, iron sulfur (Ip)

TABLE 7d DLPFC MD Genetic Ontology Genbank Accession Gene No. NameDescription transmission of nerve impulse D11428 PMP22 peripheral myelinprotein 22 AF049498 SCN2B sodium channel, voltage-gated, type II, betapolypeptide M82919 GABRB3 gamma-aminobutyric acid (GABA) A receptor,beta 3 X59834 GLUL glutamate-ammonia ligase (glutamine synthase) X81438AMPH amphiphysin (Stiff-Man syndrome with breast cancer 128 kDaautoantigen) M54927 PLP1 proteolipid protein 1 (Pelizaeus-Merzbacherdisease, spastic paraplegia 2, uncomplicated) Z11695 MAPK1mitogen-activated protein kinase 1 U01824 SLC1A2 solute carrier family 1(glial high affinity glutamate transporter), member 2 M32886 SRI sorcinU40215 SYN2 synapsin II X15376 GABRG2 gamma-aminobutyric acid (GABA) Areceptor, gamma 2 D26443 SLC1A3 solute carrier family 1 (glial highaffinity glutamate transporter), member 3 X68194 SYPL synaptophysin-likeprotein U61849 NPTX1 neuronal pentraxin I neurogenesis D11428 PMP22peripheral myelin protein 22 U30930 UGT8 UDP glycosyltransferase 8(UDP-galactose ceramide galactosyltransferase) W28770 NP25 neuronalprotein M54927 PLP1 proteolipid protein 1 (Pelizaeus-Merzbacher disease,spastic paraplegia 2, uncomplicated) D83017 NELL1 NEL-like 1 (chicken)U34846 AQP4 aquaporin 4 Z70276 FGF12 fibroblast growth factor 12 M80899AHNAK AHNAK nucleoprotein (desmoyokin) M57399 PTN pleiotrophin (heparinbinding growth factor 8, neurite growth-promoting factor 1) AF016004GPM6B glycoprotein M6B X70326 MLP MARCKS-like protein AF036268 SH3GL2SH3-domain GRB2-like 2 M93426 PTPRZ1 protein tyrosine phosphatase,receptor-type, Z polypeptide 1 U61849 NPTX1 neuronal pentraxin I M93650PAX6 paired box gene 6 (aniridia, keratitis) phosphoric ester hydralaseactivity X55740 NT5E 5′-nucleotidase, ecto (CD73) AF001601 PON2paraoxonase 2 X68277 DUSP1 dual specificity phosphatase 1 L35594 ENPP2ectonucleotide pyrophosphatase/phosphodiesterase 2 (autotaxin) AB013382DUSP6 dual specificity phosphatase 6 L05147 DUSP3 dual specificityphosphatase 3 (vaccinia virus phosphatase VH1-related) Z48541 PTPROprotein tyrosine phosphatase, receptor type, O N36638 PPP1R3C proteinphosphatase 1, regulatory (inhibitor) subunit 3C AJ001612 PSPHLphosphoserine phosphatase-like AF017786 PPAP2B phosphatidic acidphosphatase type 2B U60644 PLD3 phospholipase D3 AF056490 PDE8Aphosphodiesterase 8A M93426 PTPRZ1 protein tyrosine phosphatase,receptor-type, Z polypeptide 1

TABLE 8 Selected Potential Druggable Targets Genbank Accession Gene No.Name Target category Description AB020629 ABCA8 transporter ATP-bindingcassette, sub-family A (ABC1), member 8 X63575 ATP2B2 transporterATPase, Ca++ transporting, plasma membrane 2 X71490 ATP6V0D1 transporterATPase, H+ transporting, lysosomal 38 kDa, V0 subunit d isoform 1 S74445CRABP1 transporter cellular retinoic acid binding protein 1 M34309 ERBB3tyrosine kinase v-erb-b2 erythroblastic leukemia viral oncogene homolog3 (avian) receptor M80634 FGFR2 tyrosine kinase fibroblast growth factorreceptor 2 (bacteria-expressed kinase, (SEQ ID receptor keratinocytegrowth factor receptor, craniofacial dysostosis 1, Crouzon NO: 1)syndrome, Pfeiffer syndrome, Jackson-Weiss syndrome) M64347 FGFR3tyrosine kinase fibroblast growth factor receptor 3 (achondroplasia,thanatophoric dwarfism) receptor AC004131 GPRC5B GPCR G protein-coupledreceptor, family C, group 5, member B X77196 LAMP2 ligand for celllysosomal-associated membrane protein 2 adhesion molecule U37122 ADD3regulator of adducin 3 (gamma) kinase U66879 BAD regulator ofBCL2-antagonist of cell death protease AB007943 RAP1GA1 regulator ofRAP1, GTPase activating protein 1 kinase AF060877 RGS20 regulator ofregulator of G-protein signalling 20 GTPase AL049538 RIN2 regulator ofRas and Rab interactor 2 GTPase U58334 TP53BP2 reuglator of tumorprotein p53 binding protein, 2 protein degradation U28964 YWHAZregulator of tyrosine 3-monooxygenase/tryptophan 5-monooxygenaseactivation protein, zeta polypeptide enzyme J04046 CALM3 kinasecalmodulin 3 (phosphorylase kinase, delta) AF112471 CAMK2B kinasecalcium/calmodulin-dependent protein kinase (CaM kinase) II beta D50310CCNI regulator of cyclin I kinase U37143 CYP2J2 monooxygenase cytochromeP450, family 2, subfamily J, polypeptide 2 AB006626 HDAC4 enzyme histonedeacetylase 4 X57206 ITPKB kinase inositol 1,4,5-trisphosphate 3-kinaseB M12267 OAT enzyme ornithine aminotransferase (gyrate atrophy) Y10275PSPH enzyme phosphoserine phosphatase AJ001612 PSPHL enzymephosphoserine phosphatase-like AL080061 CLIC4 channel chlorideintracellular channel 4 L08485 GABRAS channel gamma-aminobutyric acid(GABA) A receptor, alpha 5 AF016917 GABRD channel gamma-aminobutyricacid (GABA) A receptor, delta L19182 IGFBP7 regulator of insulin-likegrowth factor binding protein 7 receptor ligand M29273 MAG myelinationmyelin associated glycoprotein X76220 MAL myelination mal, T-celldifferentiation protein Z24725 MIG2 signal mitogen inducible 2transduction AB018342 MYO10 partner for myosin X calmodulin-like proteinAB018305 SPON1 axon growth spondin 1, (f-spondin) extracellular matrixprotein guidance

1. A method for determining whether a subject is predisposed for majordepression disorder, the method comprising the steps of: (i) isolating asubject's brain tissue, wherein the brain tissue is dorsolateralprefrontal cortex tissue; (ii) contacting the subject's isolated braintissue with a nucleic acid reagent that selectively associates with apolynucleotide with 95% identity to SEQ ID NO. 1; (iii) detecting thelevel of reagent that selectively associates with the saidpolynucleotide; and (iv) comparing the detected level of selectivelyassociated reagent with a control, whereby if the detected level issignificantly less than the control, an increased likelihood that thesubject has or is predisposed for major depression disorder isdetermined; and whereby, if the detected level is not significantly lessthan the control, an increase in said likelihood is not determined bythe method. 2.-29. (canceled)
 30. The method of claim 1, wherein thesubject is deceased.